Background
==========

Mefloquine was developed by the US Army and introduced for the treatment of malaria in the late 1970s. \[[@B1]\] Mefloquine was first used for prophylaxis in 1985, and since then approximately 14.5 million people have been prescribed the drug for malaria prevention, versus 1.6 million for treatment. \[[@B2]\]

In the first decade of mefloquine\'s use, the reported adverse effects were mainly gastrointestinal.\[[@B3]\] In the late 1980s it became clear that mefloquine could cause neuropsychiatric adverse effects.\[[@B4]\] The first randomised controlled trial of mefloquine prophylaxis in heterogeneous, non-immune Western travellers was published in 2001 and found that one-third of all mefloquine users reported neuropsychiatric adverse effects and 6% of all users reported at least one severe adverse event (defined as requiring medical advice).\[[@B5]\]

On the evidence from a case series published in 1992 by the manufacturer, Hoffmann-La Roche, the World Health Organisation (WHO) recommends that travellers with a personal or family history of seizures or manic-depressive illness should not take mefloquine prophylaxis.\[[@B6],[@B7]\] However the Centers for Disease Control and their Canadian equivalent, CATMAT, do not recognise this as a valid contraindication to taking mefloquine.\[[@B8]\]

A recent analysis of spontaneous reports held on the Dutch national pharmacovigilance database suggested that there is a mefloquine syndrome consisting of excessive sweating accompanied by malaise, nausea, diarrhoea, agitation, concentration problems and nightmares.\[[@B9]\]

The aetiology of the adverse effects associated with mefloquine use remains obscure. Ten cohort studies in tourists found that women generally experienced worse side effects from mefloquine than men; \[[@B6],[@B10]-[@B18]\] an eleventh did not find this effect.\[[@B19]\] In randomised controlled trials, children have tolerated mefloquine well. \[[@B20],[@B21]\] Surprisingly, one cohort study found that some older adult travellers tolerate mefloquine better than younger adults. \[[@B22]\] It has also been reported that Asian patients tolerate mefloquine better than Caucasians and Africans. \[[@B23],[@B24]\] Despite early concerns, mefloquine appears to be safe in pregnancy.\[[@B25]\]

Although the adverse effects of mefloquine are common, and often serious and long lasting, and the drug has been widely used for over 20 years, no real attempts have so far been made to investigate and explain these effects. A systematic review of mefloquine prophylaxis, performed within the Cochrane Collaboration, now tabulates 516 case reports published in 136 papers between 1976--2000, describing adverse effects from mefloquine at prophylactic and therapeutic dosages.\[[@B26]\]

We retrieved and critically reviewed all the original papers listed in the Cochrane review to clarify the phenomenology of the adverse effects associated with mefloquine, and to look for clues to possibly safer use of the drug.

Analysis of case reports
========================

Common features of the case reports
-----------------------------------

Of the 516 published case reports of mefloquine adverse effects recorded in the mefloquine systematic review, 328 of the reports related to prophylactic mefloquine use, and 188 to treatment use. 324 of the 516 cases (63%) related to tourists or business travellers.\[[@B26]\] One-third of the reports were in languages other than English -- mainly French, German and Danish. The search strategy for finding the case reports is described in the review.\[[@B26]\] An annotated bibliography of the 516 published reports can be found at <>.

26% of the case reports recorded three or fewer individual patient parameters (for example, the patient\'s age, sex and mefloquine dose taken), and many of these reports were little more than a short description of an unexpected adverse event, set in the context of a larger study. 52% of the reports however contained some discussion of the causality of the symptoms attributed to the drug, and 11% proposed a mechanism by which these symptoms might be occurring.

56% of the 516 case reports we reviewed described one or more symptoms consistent with a *transient, anicteric chemical hepatitis* (eg, malaise, fever, anorexia, headache, abdominal pain, nausea, diarrhoea, concentration difficulties). Of the remaining reports, many were consistent with a *post-hepatic syndrome,* although in some cases the disorders described could have been due to conditions such as anxiety, depression, chronic fatigue or jet lag.

15% of all the case reports described symptomatology suggesting acutely *disturbed thyroid function* (eg, anorexia, fatigue, tremor, palpitations, nervousness, increased sweating, mood and/or sleep disturbance, memory and concentration disorders, emotional lability, altered bowel habit, depression).

Table [1](#T1){ref-type="table"} categorises the 516 published case reports according to whether the clinical features were \'very likely\', \'plausibly\' or probably \'unrelated\' to liver or thyroid pathology. We have based this classification on standard lists of the common symptoms and signs of liver and thyroid disease. \[[@B27],[@B28]\] The direction of thyroid symptoms was mainly towards hyperthyroidism, though some patients exhibited signs of both an over- and an under-active thyroid (for example, tachycardia alternating with bradycardia).

::: {#T1 .table-wrap}
::: {.caption}
######

Clinical features of published case reports of adverse effects from mefloquine, and their possible relation to liver and thyroid pathology
:::

                                                                    **Prophylaxis case reports (N = 328)**   **Treatment case reports (N = 188)**
  ----------------------------------------------------------------- ---------------------------------------- --------------------------------------
  Number (%) of reports very likely **liver** related               **75/328***(23)*                         **85/188***(45)*
  Number (%) of reports plausibly but debatably **liver** related   **102/328***(31)*                        **29/188***(15)*
  Number (%) of reports plausibly **thyroid** related               **51/328***(16)*                         **26/188***(14)*
  Probably unrelated to liver or thyroid                            **100/328***(30)*                        **48/188***(26)*
  Median dose (*range*) of mefloquine taken (mg)                    **750***(250--8750)*                     **1250***(150--5250)*
  Median duration (*range*) of adverse effects (days)               **16***(1--550)*                         **4***(1--300)*
:::

The table also shows the median duration of symptoms reported by \'prophylactic\' and \'treatment\' mefloquine users, and the median dose taken within each category. The median duration of adverse effects of patients who took mefloquine as treatment appears to have been shorter than that of those who took the drug as prophylaxis (4 days versus 16 days), even though the median dose of mefloquine taken was higher in the treatment group. We believe that one explanation for this unexpected finding may lie in the fact that the treatment users of mefloquine were more likely to have taken the drug as monotherapy, whereas the prophylaxis users more commonly took one or more co-medications, as well as alcohol. We discuss the possible significance of this later in this paper.

Mefloquine and the liver
------------------------

Mefloquine is an aryl amino alcohol which accumulates in both the liver and the lungs, and is subject to enterohepatic circulation. \[[@B29]\] It has recently been found to cause acute hepatitis.\[[@B30]\]

Mefloquine does not appear to cause florid signs of liver disease. However, transient subclinical disturbances of liver function are a common feature of many drugs metabolised in the liver, and this may explain the frequent finding of transaminase changes in safety studies of new drugs; these biochemical findings are usually dismissed as meaningless noise, but they may in fact be sensitive or oversensitive markers of vulnerability, of low specificity.

That mefloquine induces liver enzymes is well documented. Jaspers et al reported significantly raised transaminases in Dutch marines who took mefloquine during 3 months in Cambodia, and who were not drinking alcohol at the time. \[[@B31]\] Takeshima found that of a cohort of healthy Japanese soldiers who took prophylactic mefloquine for 36 weeks without drinking alcohol, one-quarter developed symptoms compatible with liver pathology and four showed disturbed liver function. \[[@B32]\] Reisinger et al observed the same phenomenon in a cohort of short-stay European travellers to Africa, but it is not clear whether alcohol could have contributed to this effect.\[[@B33]\] One of the travellers, who was concurrently taking a liver-damaging agent, sulfadoxine,\[[@B34]\] showed gross morphological changes in his liver which were attributed to his use of prophylactic mefloquine. Liver biopsy showed intralobular cellular infiltrates consisting of macrophages and eosinophils as well as sporadic eosinophilic cell necroses; virology was negative. \[[@B33]\] Grieco et al described a 46-year-old woman who drank wine daily while taking mefloquine, and who became nervous and depressed, with nausea, vomiting and diarrhoea. She was dehydrated and in severe liver failure, with negative virology. Liver biopsy showed diffuse macrovesical hepatic steatosis. \[[@B35]\]

\'Heavy sun exposure\' is noted in a case report of a 60-year-old Frenchman who reacted acutely to his second mefloquine tablet; it is likely that this sun exposure would have caused dehydration.\[[@B5]\] A 20-year old French traveller, concurrently taking an oral contraceptive, had epileptic seizures in her sixth week of mefloquine prophylaxis, directly after \'severe exertion\'.\[[@B5]\] It seems likely that in some mefloquine users dehydration will impose an added burden on the liver, and that this could contribute to a severe reaction to the drug. Many long-haul travellers using mefloquine are mildly dehydrated from in-flight alcohol and air conditioning, followed by hot and dry conditions, and more alcohol consumption, at their holiday or business destination.

Of the 516 case reports we reviewed, eleven cited alcohol as possibly contributing to the adverse drug effects described. Wittes et al reported a remarkable challenge-rechallenge experiment where a healthy male geologist took both his third and his fourth weekly mefloquine tablet together with half a bottle of whisky, and on both occasions experienced acute paranoid delusions, depression and suicidal ideation; a fellow geologist who shared the same whisky bottle (and who was taking no antimalaria medication) experienced no such effects.\[[@B36]\]

Vuurman et al, sponsored by Hoffmann-La Roche, tested in healthy volunteers whether or not alcohol might interact adversely with mefloquine.\[[@B37]\] They found psychomotor performance unimpaired, but their study design had important limitations. Only 20 participants took mefloquine and of these, two women dropped out due to adverse events (one with nausea, vomiting and dizziness, the other with malaise, fever and headache). The study protocol forbade \'strenuous physical activity\' and any prescribed medications. Alcohol was given under strict laboratory conditions 24 h after mefloquine ingestion, and then in small and interrupted doses, such that the blood alcohol concentration in any participant never exceeded 0.50 mg ml^-1^. The authors admit that their study did not address \'the question of what might happen should (mefloquine users) consume intoxicating amounts of alcohol. \[[@B37]\] Their findings can thus not be generalised to the broad population of tourists and business travellers.

Approximately half of the case reports listed in the Cochrane review note some co-medication taken along with mefloquine.\[[@B26]\] Other quinoline derivatives (chiefly chloroquine and quinine) are the commonest co-medications mentioned in the case reports. After antimalaria drugs, an oral contraceptive (noted in 8 reports) is the next most commonly reported co-medication, followed by sodium valproate (7) and diazepam (4). All these drugs can cause liver damage.\[[@B34]\] Diazepam is also a thyroid hormone antagonist, and we discuss below the possible significance of this. Meszaros et al reported a male traveller who in addition to mefloquine took thioridazine, amitriptyline and fluphenazine (all capable of damaging the liver), and whose mefloquine-associated neuropsychiatric symptoms persisted for over a year. \[[@B38]\] Gullahorn et al reported a series of patients who experienced delirium on emerging from anaesthesia, possibly because in addition to mefloquine they had received isoflurane, an anaesthetic known to cause hepatocellular necrosis. \[[@B34],[@B39]\]

One report describes an acute reaction in a man who took one mefloquine tablet each week together with two aspirin tablets. One hour after taking his fifth mefloquine tablet he experienced acute amnesia lasting approximately one hour.\[[@B40]\] Aspirin can cause hepatocellular necrosis,\[[@B34]\] and in addition can aggravate acute thyroid disturbance (discussed below) by competing with thyroid hormones for sites on binding proteins.\[[@B53]\]

Mefloquine and the thyroid
--------------------------

The preclinical studies of mefloquine by the US Army involved close monitoring in animal models and human volunteers of several organ systems, but not the thyroid. \[[@B1]\] The effect of mefloquine on thyroid function appears not to have been investigated in any phase III or phase IV study. Thyroid function has not been tested routinely in the diagnosis or management of patients suffering from mefloquine-related adverse effects.

Thyroid disease, or some possible interference with thyroid activity, is reported in only three of the 516 case reports in the Cochrane review.\[[@B26]\] Bem et al described a 31-year old German woman with a \'thyroid condition\', who was also taking \'tranquillisers\' (unspecified) and alcohol, and who had an acute exacerbation of her schizophrenia after a single tablet of mefloquine; her symptoms persisted for 4 weeks.\[[@B5]\]Conget et al reported a 30-year old previously healthy woman who experienced abdominal pain, palpitations, instability, insomnia and a fine distal tremor in her second week of mefloquine prophylaxis. A thyroid function screen showed a raised serum thyroglobulin level (54 μg/ml, normal range 18.7 to 27.1); this returned to normal within a month of her stopping mefloquine.\[[@B41]\] Bauer et al reported acute psychotic reactions in a healthy US Peace Corps worker who took prophylactic mefloquine concurrently with diiodohydroxyquinoline for a presumed parasitic infection.\[[@B42]\]

Presentation of the hypothesis
==============================

The mefloquine syndrome
-----------------------

The published literature describes a mefloquine syndrome that presents in a variety of ways including headache, gastrointestinal disturbances, nervousness, fatigue, disorders of sleep, mood, memory and concentration, and occasionally frank psychosis. Young western women are particularly vulnerable to mefloquine\'s adverse effects. Certain groups however (children, older adult travellers and Asian patients) tolerate mefloquine well.

Hypothesis
----------

The phenomenology of mefloquine\'s adverse effect profile, together with incidental details in some of the published case reports (references to alcohol, and to known hepatotoxic co-medications such as the oral contraceptive pill) suggest that for many mefloquine users adverse drug effects may be the result of primary hepatocellular injury, caused by the drug in association with one or more concurrent liver insults. Further, it seems that in some of these symptomatic users of mefloquine a transient thyroid disturbance may appear as well, either as an endocrine disorder secondary to the primary liver damage, or as an independent pathological process.

We therefore postulate that many of the adverse effects of mefloquine are a post-hepatic syndrome caused by primary liver damage. In some individuals we believe that symptomatic thyroid disturbance occurs, either as an independent process, or as a secondary consequence of the initial hepatocellular injury.

Previous liver or thyroid disease, and concurrent insults to the liver (such as from alcohol, dehydration, an oral contraceptive pill, recreational drugs, and other drugs that can damage the liver) may be related to the development of many severe or prolonged adverse reactions to mefloquine. Co-medications that are thyroid hormone antagonists may also be risk factors.

Relevant reports with other quinoline derivatives
-------------------------------------------------

Mefloquine is a synthetic quinoline; other quinolines include primaquine, amodiaquine and chloroquine.\[[@B43]\]

High doses of primaquine in rhesus monkeys have caused acute fatal liver damage.\[[@B44]\]

Amodiaquine is still used to treat malaria, but was withdrawn from general use for malaria prophylaxis in 1986 after it was found to cause liver damage and hepatitis, mostly anicteric. \[[@B45],[@B50]\] Some of these reports mention co-medications known to damage liver cells (phenylbutazone, oral contraceptive, alcohol). \[[@B45],[@B46]\] Amodiaquine has not been linked to disturbed thyroid function.

The possibility of a three-way interaction has already been suggested between a quinoline derivative (chloroquine) and the liver and the thyroid. Munera et al described a woman with hypothyroidism, well stabilised on thyroxine sodium 125 μg daily, who took prophylactic chloroquine and proguanil daily for 2 months for a vacation in Africa.\[[@B51]\] At four weeks her thyroid stimulating hormone (TSH) concentration was found to be very high (44.8 mU/1, normal range 0.35--6.0), but it returned to normal within a week of her stopping the drugs. Re-challenge with chloroquine and proguanil a year later again resulted in raised levels of TSH, a lowered concentration of free triiodothyronine (T~3~), and normal free thyroxine (T~4~) concentration. Liver function was not tested, but the authors speculated that \'Chloroquine\... seems to have enhanced the induction of liver enzymes. \[It\] probably increased the catabolism of thyroid hormones by enzymatic induction.\[[@B51]\] They also suggested that chloroquine might act centrally on the hypothalamus, through disruption of the feedback system by which thyrotropin releasing hormone stimulates the pituitary to release and later synthesise TSH.\[[@B52]\]

A third mechanism by which chloroquine and chemically related drugs such as mefloquine might interfere with thyroid function is through structural homology to T~3~, resulting in thyroid hormone antagonism.\[[@B53]\] In the rat, chloroquine injections more than halve the T~3~ concentration, without changing the level of free T~4~.\[[@B54]\] Chloroquine has been reported to inhibit T~3~ uptake in mammalian cells by inhibiting receptor-mediated endocytosis. \[[@B53]\]

Testing the hypothesis
======================

Our hypothesis needs to be tested through a large multicentre cohort study of mefloquine prophylaxis in tourists and business travellers, perhaps recruited in collaboration with one or more airlines. Small randomised controlled trials should test specific elements of the hypothesis, such as the postulated link between mefloquine and thyroid disturbance, and the presumed interaction between mefloquine and oral contraception. National pharmacovigilance databases should also be analysed systematically to see if the spontaneous reports of mefloquine\'s adverse effects tend to support our hypothesis or not.

The multicentre cohort study of prophylactic mefloquine use should be questionnaire-based, and should enquire specifically into the major risk factors (alcohol intake during travel, hydration status, use of hormonal contraception and recreational drugs, other potentially hepatotoxic or thyrotoxic drugs, previous history of proven or suspected liver and/or thyroid abnormality) that we have proposed. The study design should include pre- and post-exposure testing of liver and thyroid function in at least a sample of the cohort.

One or more case-control studies should be nested within the cohort study. \[[@B55],[@B56]\] These nested studies would allow for rigorous testing of the aetiological mechanisms which we have proposed for mefloquine\'s adverse effects. The studies should also resolve those prescribing issues on which experts\' opinions differ (eg, Is mefloquine safe in pregnancy? Is it safe for long-term prophylaxis? Should airline pilots be prescribed mefloquine? Should mefloquine be prescribed to people with a personal or family history of neuropsychiatric illness? Can mefloquine be given safely as a pre-travel loading dose, for rapid induction of chemoprophylaxis? \[[@B17],[@B57]\]).

Mefloquine is a clinically important drug, commonly used by healthy people. A much higher standard of safety is therefore required for mefloquine prophylaxis than for drugs given to treat serious diseases.\[[@B58],[@B59]\] The study we propose is urgently needed.

Implications of the hypothesis
==============================

What our hypothesis explains
----------------------------

The hypothesis explains much of the complexity of the pattern of adverse effects of mefloquine, and also the fact that many healthy users of the drug suffer no adverse effects at all. It also explains some aspects of mefloquine\'s tolerability profile in travellers which until now have not been understood, notably that young women experience more adverse effects from mefloquine than men, and that children and older adult travellers (who do not use oral contraception, and who rarely misuse alcohol) seem to tolerate the drug well. In addition, our hypothesis may explain the ethnic and inter individual variations in the pharmacokinetics of this agent, which until now have not been understood. \[[@B23],[@B24]\]

It has been known for some years that mefloquine users who take co-medications are about 1.5 times more likely to experience an adverse drug event than those users who take no co-medications, and twice as likely to experience severe adverse drug events.\[[@B14]\] The frequency of reported adverse drug events also increases when multiple co-medications are taken.\[[@B14]\] Our hypothesis plausibly explains these earlier findings.

The use of marijuana and other psychoactive agents by chloroquine users has been associated with acute psychotic reactions, and it has been suggested that mefloquine users who take recreational drugs may likewise be predisposed to neuropsychiatric problems.\[[@B60],[@B61]\] We believe that this association is consistent with our hypothesis, since recreational drugs can cause hepatocellular damage and liver failure.\[[@B34]\]

A puzzling observation is that although the adverse effects of mefloquine are usually reversible, in some patients these effects can persist for months or even years after the drug has been stopped.\[[@B61]\] We believe that the occasionally protracted time course of the adverse effects can be plausibly explained by supposing that in these patients mefloquine is just one of several concurrent insults to the liver, and that it is the continuance of the other insults (most commonly alcohol, and certain prescription drugs) that makes the mefloquine-induced syndrome persist. Some published evidence supports this view.\[[@B38],[@B62],[@B69]\] There is evidence from this study (Table [1](#T1){ref-type="table"}) that the adverse effects of mefloquine in those who have taken the drug prophylactically persist longer than they do in patients who have been treated with it, even though the latter group mostly receive larger doses of the drug; this paradox might be explained by the fact that although prophylactic users usually stop mefloquine as soon as they have experienced adverse effects, they often continue to assault their livers in other ways, and it is this which makes the effects persist.

Most of the reported adverse effects of mefloquine fit into the model we propose. For example, mefloquine has been associated with a wide variety of dermatological adverse effects, and most of these can be linked with effects on the liver or thyroid.\[[@B41],[@B55]\] Convulsions and dizziness are other reported effects of mefloquine which can be related to liver or thyroid disturbance.\[[@B2],[@B41]\]

Who should not take mefloquine?
-------------------------------

We believe that people with a history of any proven or suspected liver or thyroid abnormality in the previous two years should avoid mefloquine. Travellers taking mefloquine should not drink alcohol, especially within 24 h of their weekly mefloquine dose.

While taking mefloquine, travellers should be advised to maintain good hydration with water or carbonated drinks, especially on long plane journeys or during arduous work in hot conditions. Alcohol, tea or coffee should not be used to maintain hydration, since they all increase water loss.

Travellers taking mefloquine should not take a hormonal contraceptive, nor any other drug known to injure liver cells.\[[@B34]\] They should not take any drug known to antagonise thyroid hormone. \[[@B53]\] We propose that drugs that are known to cause hepatocellular injury *and also* to be thyroid hormone antagonists should be absolutely contraindicated in mefloquine users; such drugs include amiodarone, benzodiazepines, calcium channel blockers and phenytoin.\[[@B34],[@B53]\]

Because of the potential for additive toxicity, travellers taking mefloquine should avoid concurrent use of any other quinoline derivative (eg, amodiaquine, chloroquine, primaquine, quinidine, quinine, tafenoquine), whether for additional prophylaxis or for treatment. The administration of a different quinoline derivative at the same time as mefloquine may increase the risk of adverse effects.\[[@B70],[@B72]\] For the same reason, mefloquine users should avoid other quinine analogues, such as fluoroquinolone antibiotics. Fluoroquinolones, such as sparfloxacin, of loxacin and ciprofloxacin, are increasingly prescribed in severe cases of traveller\'s diarrhoea, and have been associated with severe reactions in mefloquine users. \[[@B73]\]

Who should take mefloquine?
---------------------------

Mefloquine is a safe and exceptionally useful drug for the mass prophylaxis and treatment of those resident populations in malaria-endemic areas which traditionally abstain from alcohol and hormonal contraception. It has been suggested that in such settings, mefloquine should be combined with artemisinin or a derivative to protect both drugs from resistance.\[[@B74]\]

On the basis of our hypothesis, we believe that children, and also pregnant women, can safely be prescribed mefloquine (because neither group uses alcohol or oral contraceptives).

Accompanied by explicit advice to avoid alcohol, maintain good hydration, and use co-medications with caution, we believe that prophylactic mefloquine could be recommended to certain occupational subsets of travellers who carry out safety-critical tasks and who until now have been denied the use of this drug. These occupational groups include airline pilots,\[[@B75]\] divers\[[@B76]\] and operators of heavy machinery.\[[@B77]\]

Some authorities advise that travellers engaged in high-risk leisure pursuits, such as mountain climbing, should not use mefloquine. \[[@B78]\] We believe that this exclusion is unjustified, as long as there is no recent history of liver or thyroid disease, and provided the precautions we have proposed above (avoidance of alcohol, maintenance of hydration and non-use of hormonal contraception, recreational drugs and certain co-medications) are adhered to.

Lobel et al consider that WHO\'s exclusion of people with a personal or family history of neuropsychiatric illness from taking mefloquine is based \'on limited evidence or theoretical concerns\', and we believe their scepticism is justified.\[[@B79]\] Neuropsychiatric illness may not contraindicate use of mefloquine, provided that the patient is not currently taking anything that can cause liver damage or thyroid disturbance.

Competing interests
===================

None declared.

::: {#T2 .table-wrap}
::: {.caption}
######

Proposed contraindications to the use of mefloquine
:::

  ----------------------------------- -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
  **Mefloquine contraindications:**
  1\.                                 Known **hypersensitivity** to the drug.
  2\.                                 History of any proven or suspected **liver abnormality** within the previous 2 years.
  3\.                                 History of any proven or suspected **thyroid abnormality** within the previous 2 years, including any concurrent use of thyroid-stabilising medication.
  4\.                                 Concurrent use of drugs known to cause **hepatocellular injury.** Paracetamol and (especially) aspirin to be used with caution, since both can damage the liver and/or the thyroid.\[[@B34],[@B40]\] Mefloquine users should not take recreational drugs.
  5\.                                 Concurrent use of **oral contraceptive pill** or **HRT.** Healthcare advisers should recognise that some female travellers will need alternative contraception.
  6\.                                 Concurrent use of drugs known to be **thyroid hormone antagonists.**\[[@B53]\]
  7\.                                 Up to 2 units of **alcohol** per day may be taken by users of mefloquine prophylaxis until 24 h before their weekly dose, and from 24 h afterwards.
  8\.                                 Mefloquine users should avoid concurrent use of **any other quinoline derivative** (eg, amodiaquine, chloroquine, primaquine, quinidine, quinine, tafenoquine), whether for additional prophylaxis or for treatment.
  ----------------------------------- -----------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------------
:::

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Acknowledgements
================

We thank Professor Alan McGregor FRCP, of Guy\'s, King\'s and St Thomas\' School of Medicine, London, for his helpful comments on an earlier draft of this paper.


Background
==========

Pleural effusion is a common clinical problem resulting from thoracic or systemic diseases. An effusion is termed as transudate when its formation is due to alterations in the mechanical factors such as increased pulmonary hydrostatic pressure or decreased plasma oncotic pressure \[[@B1],[@B2]\]. An effusion is classified as an exudate when the pleural fluid is formed because of loss of pleural surface integrity by inflammatory or infiltrative processes causing an increase in microvascular permeability \[[@B1],[@B3]\].

Based on this principle pleural fluid is termed an exudate if fluid to serum total protein ratio (TPR) is ≥ 0.5, or the pleural fluid absolute value of FLDH is ≥ 200 IU/L, or the fluid to serum ratio of LDH value (LDHR) is ≥ 0.6. An effusion is classified as a transudate if the TPR is \<0.5, FLDH is \<200 IU/L, and the LDHR is \<0.6 \[[@B4]\]. Roth and associates have documented that serum-effusion gradient of albumin is a better discriminator than Light\'s criteria in the diagnostic separation of transudates and exudates \[[@B5]\]. On the other hand, Burgess *et. al.* \[[@B6]\] using an albumin gradient of 12 g/L found the sensitivity and specificity to be 87% and 92% respectively and concluded that the criteria by Light *et. al* \[[@B4]\] remained the best method for distinguishing exudates from transudates. It is worth noting here that both studies used conventional analysis and not Receiver Operating Characteristics (ROC).

Recently, we have established that FLDH and TPR perform better and that fluid to serum ratio of LDH (LDHR) had no role in the diagnostic separation of transudates and exudates using ROC analysis \[[@B7]\]. Therefore, this study was undertaken to compare the accuracy of serum-effusion albumin gradient (SEAG) and fluid to serum albumin ratio (ALBR) with the best available criteria i.e. FLDH from our previous report using ROC \[[@B7]\].

Materials & Methods
===================

Patients referred for evaluation of pleural effusion to the respiratory unit at Rotherham Trust hospital, United Kingdom, from January 1989 to June 1991 for a prospective investigation of the dynamics of pleural effusion formation and removal were included in the study \[[@B8]\]. The cause of pleural effusion and their diagnostic separation was determined using clinical criteria set by Light and associates \[[@B4]\]. All patients were followed for at least 3 months or till the final cause of the pleural effusion was established. During the study, pleural effusion samples from two hundred and twelve (212) consecutive patients were collected. However, eight patients with uncertain diagnosis and four patients with possible multiple causes for the pleural effusion were excluded from the analysis. Effusions due to pulmonary embolism were classified as exudates according to Burgess *et al* \[[@B6]\].

Blood and pleural effusion samples collected and stored at -80°C were analyzed within a period of six months for albumin, total protein and LDH level. The pleural fluid and the corresponding serum samples were drawn simultaneously at the time of admission before any treatment was administered. LDH was measured with Boehringer Mannheim kit according to established methods and results expressed in IU/L \[[@B9]\]. The upper limit for the normal serum LDH value in our laboratory during the study was 200 IU/L. SEAG was calculated by subtracting the pleural fluid albumin value from serum albumin value.

Statistical analysis
--------------------

The median values for SEAG, ALBR and FLDH were compared in transudates and exudates using Mann-Whitney U test. A p-value of \<0.05 was considered significant. To compare the performance of SEAG, ALBR and FLDH in the diagnostic separation of transudates and exudates, ROC curves were generated for each of the criteria by plotting the true positive value (sensitivity) against the false positive value (1- specificity) for multiple test results. Area under the curve (AUC) with 95% confidence intervals was calculated using Wilcoxon estimate. The AUC for the three tests were compared using the method described by Hanley and McNeil \[[@B10]\]. Furthermore, to account for multiple comparisons, we performed Bonferoni\'s correction of the final p value. Optimum cut-off points for SEAG, ALBR and FLDH were computed by using the statistical software StatsDirect () taking the best possible values for sensitivity and 1-specificity on the ROC plot. Finally, the correlation between SEAG and ALBR was estimated using Pearson\'s correlation. Data on age is presented as mean ± standard deviation and other test variables are presented as median and interquartile range (IQR) as they are not distributed normally.

Results
=======

Of the 200 patients with pleural effusion, 116 were males and 84 were females. The mean age ± standard deviation was 62 ± 15. One hundred and fifty six (78%) effusions were exudates. The causes for the pleural effusion were: thoracic malignancy 90, thoracic infection including empyema 37, pulmonary embolism 11, pancreatitis 6, autoimmune disease 5, other exudates 7, cardiac failure 37, chronic liver disease 5, and nephrotic syndrome 2. The median and IQR for SEAG, ALBR and FLDH are given in table [1](#T1){ref-type="table"}. The median value for all three tests in transudates and exudates were significantly different.

::: {#T1 .table-wrap}
::: {.caption}
######

Median and Interquartile range (IQR) for the three tests
:::

  **Test**      **Transudate (n = 44)**   **IQR**       **Exudate (n = 156)**   **IQR**      **P value ^\*^**
  ------------- ------------------------- ------------- ----------------------- ------------ ------------------
  SEAG (g/L)    93.5                      33.8--122.5   308.5                   171--692     0.0001
  ALBR          0.49                      0.42--0.62    0.77                    0.63--0.85   0.0001
  FLDH (IU/L)   98.5                      76.8--127.5   344                     216--695     0.0001

^\*^ Mann-Whitney U test
:::

The ROC plots for SEAG, ALBR and FLDH are shown in figure-[1](#F1){ref-type="fig"}. At all test values, FLDH was more sensitive and specific than SEAG and ALBR. The optimum cut-off levels were 163IU/L for FLDH, 9 g/L for SEAG, and 0.68 for ALBR respectively. FLDH was the best of the three tests. The AUC and the corresponding 95% Cl for SEAG, ALBR and FLDH were 0.81 (0.75 to 0.87), 0.79 (0.72 to 0.86) and 0.9 (0.87 to 0.96) respectively. Although the AUC for SEAG and ALBR were of similar magnitude, the AUC for FLDH was significantly more than the AUC for ALBR (z = 2.75, p \< 0.01). Of the 156 exudates, FLDH correctly classified 132 (85%) 95% Cl 0.78--0.90, SEAG 112 (72%) 0.64--0.79, and ALBR 111 (71%) 0.63--0.78 respectively as exudates and all three tests classified 39 (89%) 0.75--0.96 of the 44 transudates correctly. Table [2](#T2){ref-type="table"} lists the positive likelihood ratios (LR +ve) with 95% Cl for the three tests at the optimum cut-off level. Figure-[2](#F2){ref-type="fig"} shows the correlation between SEAG and ALBR. There was a significant negative correlation between the two tests (r = -0.89, p \< 0.0001).

![Receiver Operating Characteristic plots of the pleural fluid FLDH, ALBR, and SEAG. The optimum cut-off level was determined by selecting the points of test values that provided the greatest sum of sensitivity and specificity. The optimum cutoff for FLDH was 163 IU/L, ALBR = 0.68, SEAG = 9.0.](1471-2466-2-1-1){#F1}

![Association between ALBR and SEAG in pleural effusion](1471-2466-2-1-2){#F2}

::: {#T2 .table-wrap}
::: {.caption}
######

Positive Likelihood Ratios (LR) at optimum cut-off points
:::

  **Test**   **LR**   **95% CI**
  ---------- -------- ------------
  FLDH       7.3      3.5 to 17
  SEAG       6.3      3 to 15
  ALBR       6.2      3 to 14
:::

Discussion
==========

Pleural effusion is a common clinical problem and it\'s diagnostic approach has been simplified by characterizing effusion into transudate and exudate \[[@B11]\]. Several biochemical markers have been used in the past for this purpose \[[@B12]-[@B14]\]. However, TPR and FLDH have remained the most widely used criteria so far. Therefore, this study was undertaken to assess the global performance of SEAG and ALBR with the existing best criteria i.e. FLDH \[[@B7]\]. The strength of our study is that we have utilized ROC analysis for comparing different tests. Furthermore, ROC analysis provides an opportunity to calculate likelihood ratios at various cutoff points of the diagnostic test. The upper limit of the normal LDH varies according to the methods used for estimating lactate dehydrogenase. In this study we have used the Boehringer Mannheim kit and the upper normal for this method is 200IU//L \[[@B9]\].

In our present study, the mean age and causes of pleural effusion were similar to that of Roth et al. \[[@B5]\]. However, the mean SEAG value of 14 g/L for transudate and 8 g/L for exudate in our series were different from that provided by Roth and associates about 20 g/L for transudates and 0.75 g/L for exudates. From ROC analysis it is clear that FLDH performed better compared to SEAG and ALBR as documented by the highest value for the AUC. ALBR has been reported to be inferior to SEAG in the diagnostic separation of transudates and exudates \[[@B5]\]. However, in our analysis the accuracy of ALBR and SEAG were similar as evidenced by similar AUC and positive LR. Furthermore, correlation analysis between SEAG and ALBR has shown a significant negative correlation (0.89).

As the level of pleural fluid albumin increases depending on the degree of pleural microvacular injury in exudative processes, the ALBR increases correspondingly. Whereas, pleural fluid to SEAG is highest in transustive effusions as less albumin is filtered through the relatively normal pleural microvasculature. However, as pleural microvasculature is damaged in exudative processes, the level of albumin leaking into the pleural cavity would progressively increase depending on the degree of injury. Thus a very low SEAG can be expected in a trasudative processes with a correspondingly higher ALBR. This would partly explain the significant negative correlation observed between SEAG and ALBR thus indicating that these two tests likely to be operating through the same mechanism \[[@B15]\].

In our study using 9 g/L as the optimum cutoff level for SEAG, 51 of the 156 exudates were incorrectly labeled as transudates (false negative) and 5 of the 44 transudates were labeled as exudates (false positives). There were no trends for any specific diseases clustering in either of the groups.

The pathophysiology of transudative or exudative pleural effusion in humans is not well studied. We have previously documented that the rate of formation and absorption are different in different disease states causing pleural effusion \[[@B8]\]. The decreased concentration of total protein and albumin in transudates can be explained by the selective sieving of these macromolecules by the normal intact microvasculature in transudates \[[@B3]\]. However, animal and human studies have shown that increase in hydrostatic pressure can increase the protein content in effusions \[[@B16],[@B17]\]. This is partly explained by the opening up of large pores in endothelium with elevated hydrostatic pressure \[[@B18],[@B19]\]. Thus in a patient with pleural effusion from congestive heart failure, it is possible that the amount of protein in the pleural fluid can increase significantly to exudative levels if the degree of pulmonary artery pressure causing the formation of effusion was very high. Such a linear correlation between the hydrostatic pressure and the protein content of ascites in patients with portal hypertension has been documented \[[@B17]\]. Therefore, this phenomenon may help explain the pathogenesis of exudative effusions documented in congestive heart failure when total protein in pleural effusion was used as a diagnostic criteria \[[@B5],[@B20],[@B21]\].

The dynamics of LDH in the pleural cavity is not well studied. Our recent investigation into the dynamics of pleural fluid LDH has documented that there was no correlation between serum and pleural fluid LDH concentration in transudates or exudates \[[@B7]\]. This compartmentalized elevation of LDH concentration in pleural cavity in exudative processes help explain the superior performance of FLDH as a diagnostic test in the separation of pleural effusion into transudate and exudate compared to SEAG, ALBR. We have reported earlier that LDHR has no role in the diagnostic separation of transudates and exudates. Furthermore, FLDH is the most accurate marker and combining TPR with FLDH improves the diagnostic utility marginally \[[@B7]\].

Conclusion
==========

The discriminative value for SEAG and ALBR appears to be similar in the diagnostic separation of transudates and exudates. FLDH is a superior test compared to SEAG and ALBR. Based on these results, SEAG and ALBR appear to have limited diagnostic utility in clinical practice.

Competing interests
===================

None declared

Author contributorship
======================

Author one JJ conceived the idea, designed the study, collected the data, took part in analysis of data and drafted the manuscript.

Author two PB analyzed the data, interpreted the results and contributed in writing the manuscript.

Author three GSB designed the study, supervised the data collection and contributed in writing the manuscript.

Author four SS participated in the data analysis, interpreting results and contributed in writing and editing the manuscript.

All authors read and approved the final manuscript.

Pre-publication history
=======================

The pre-publication history for this paper can be accessed here:

<>

Acknowledgement
===============

The authors would like to thank Mr. Steve Viney for carrying out the pleural fluid biochemical analysis.


Background
==========

The genus *Hapalemur* of the Malagasy Prosimian family Lemuridae comprises three clearly different species: *Hapalemur griseus*\[[@B1]\], *H. simus*\[[@B2]\] and *H. aureus*\[[@B3]\]. Classification of different types within the *H. griseus* group has been controversial, particularly in the light of the variations existing in coat colour \[[@B4]\]. Systematic cytogenetic studies led to the description of four subspecies of *H. griseus* characterised by different karyotypes: *H. g. griseus* (HGG) (2N = 54) \[[@B5],[@B6]\], initially considered as the most widespread and supposed to live in the Eastern forest of Madagascar from the north to the south \[[@B7]\]; *H. g. occidentalis* (HGO) (2N = 58) \[[@B6]\] restricted in the north-west and the eastern part of Madagascar as east as Maroantsetra \[[@B4],[@B8]\]; *H. g. meridionalis* (HGM) (2N = 54) \[[@B9]\] found in the south, near Fort Dauphin, and differing from *H. g. griseus* in one metacentric chromosome ; *H. g. alaotrensis* (HGA) (2N = 54) \[[@B6]\], restricted to the bed of the Alaotra lake, with a karyotype similar to HGG containing more juxtacentromeric heterochromatin, and characterized by a larger body size \[[@B4]\]. In addition, a new variant of HG, *H. griseus ssp* (HGssp), has recently been described. This type is characterized by a chromosomal polymorphism (2N = 54, 55 and 56) and is living in the area of Ranomafana-Kianjavato \[[@B8]\].

The comparison of the karyotypes of the different *Hapalemur* led to a phylogenetic tree characterised by an earlier emergence *of H. simus* (HSI), followed by *H. aureus* (HAU) which represents the sister clade of the different groups of the *H. griseus* subspecies \[[@B8],[@B10]\]. As molecular studies were fruitfully used for taxonomic investigations of several taxa, including lemurs \[[@B11]-[@B15]\], and as no large morphological differences could be found between the cytogenetically close subspecies of *H. griseus,* mitochondrial cytochrome b and 12S DNA analyses were performed in order to determinate more accurately their species and/or subspecies status.

Results
=======

A total of 115 animals covering all the genus *Hapalemur* were captured in bamboo forests extending from the north to the south of Madagascar. The different capture areas of each species and subspecies as the number of animals are indicated in Fig. [1](#F1){ref-type="fig"}.

![Map of Madagascar showing the capture locations and/or the origin of the different *Hapalemur* species and subspecies, a) Analamera, b) Ambato, c) Ambakoany, d) Maroantsetra, e) Alaotra lake, f) Maromiza, g) Tsimbazaza zoo, h) Ranomafana, i) AmbolomavoJ) Kianjavato, k) Andohahela, l) Mandena. Abbreviations : HSI *= Hapalemur simus,* HAU *= H. aureus,* HGM *= H. griseus meridionalis,* HGssp~b~*= H. griseus ssp*~*b*~*,* HGssp~a~*= H. griseus ssp*~*a*~*,* HGG *= H. g. griseus,* HGA *= H. g. alaotrensis,* HGO *= H. g. occidentalis.* The first number behind each taxon represents the number of animals captured and the second, the number of haplotype found in this area. ^\*^The haplotype HGssp~a~02 is present in h, i and j; Hgssp~a~03 in h and j; Hgssp~a~07 in h and i; HGssp~a~10 in h and i.](1471-2148-2-4-1){#F1}

The PCR amplification of the cytochrome b resulted in a 357 bp fragment. Alignments using blast search demonstrated that this fragment corresponds to the beginning of the mitochondrial cytochrome b gene. All the sequences were aligned and the substitutions were scored. Animals sharing the same sequences were regrouped into a same haplotype. These different haplotypes are summarised in Fig. [1](#F1){ref-type="fig"} and table [2](#T2){ref-type="table"}. Consensus sequences constructed with the most common sequence in each species and subspecies (not shown) demonstrated that transition substitutions occur more frequently than transversions (an average of eight transitions for 1 transversion) and gaps are absent. As expected \[[@B16]\], most of the substitutions occur on the third position of the codon (54 out of 64 substitutions), while the first position changed nine times and the second once. The portion amplified comprises the four invariant codons considered necessary for cytochrome b function \[[@B17]\]. Changes were sometimes noticed in the third base of the codon in these positions, but the amino acid remains the same so that the functionality of the cytochrome b is not affected.

::: {#T1 .table-wrap}
::: {.caption}
######

Lower and higher values of Kimura two parameters distances (under the diagonal) and absolute distances (above the diagonal) between haplotypes of *Hapalemur* species and subspecies. For abbreviations see Figure [1](#F1){ref-type="fig"}.
:::

           HSI                   HAU                   HGM                   HGsspb                HGA                   HGG                   HGsspa                 HGO
  -------- --------------------- --------------------- --------------------- --------------------- --------------------- --------------------- ---------------------- ---------------------
  HSI      **0--7 0--0.01991**   36 42                 38 46                 38 42                 30 35                 30 36                 30 39                  31 40
  HAU      0.11024 0.13002       **0--2 0--0.00563**   41 48                 42 46                 31 34                 30 36                 30 36                  34 39
  HGM      0.11797 0.14524       0.12893 0.15338       **0--9 0--0.02565**   0 10                  23 28                 21 29                 21 34                  25 32
  HGsspb   0.11797 0.13085       0.13290 0.14524       0.00000 0.02855       **0--5 0--0.01414**   22 26                 21 27                 21 32                  25 30
  HGA      0.09098 0.11075       0.09431 0.10393       0.06558 0.08459       0.06558 0.07747       **0--1 0--0.00281**   1 3                   2 9                    4 10
  HGG      0.09098 0.11075       0.09098 0.11075       0.06240 0.08789       0.06240 0.08092       0.00281 0.00847       **0--4 0--0.01133**   3 10                   5 11
  HGsspa   0.09098 0.12087       0.09098 0.11049       0.06240 0.10473       0.06240 0.09719       0.00563 0.02580       0.00847 0.02875       **0--10 0--0.02868**   5 13
  HGO      0.09431 0.12469       0.10445 0.12117       0.07522 0.09767       0.07522 0.09055       0.01133 0.02883       0.01421 0.03180       0.01421 0.03781        **0--6 0--0.01705**
:::

Kimura two parameter distances \[[@B18]\] and absolute distances calculated with the different haplotypes (Table [1](#T1){ref-type="table"}) as phylogenetic trees constructed with the computer program Pylogenetic Analysis Using Parsimony (PAUP) 4.0b.4a \[[@B19]\] (Figs [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}, [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}) were used to compare the different *Hapalemur* species and subspecies. Neighbor-joining analyses of cytochrome b haplotypes demonstrated that *H. simus* emerged first followed by *H. aureus* which is the sister clade of the *H. griseus* subspecies. Inside *H. griseus* subspecies, two subclades are present and the *H. griseus ssp* (HGssp) are distributed in both the subclades (Figs [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}). For this reason, the *H. griseus ssp* were separated in HGssp~a~ and Hgssp~b~. The first subclade (S1) contains HGA, HGG, HGssp~a~ and HGO. The second subclade (S2) is composed of HGM and HGssp~b~. Maximum parsimony analyses (data not shown) demonstrated the same topology, unless the bootstrap support for S1 dropped from 94 to 66.

![Phylogenetic tree based on 357 bp sequences of the cytochrome b gene. Bootstrap method with neighbor joining search and Kimura two parameter distance correction obtained with 10000 replications are used. Only nodes with a bootstrap value greater than 50% are indicated. The tree is rooted using an *Eulemur macaco flavifrons* (EMF) sequence and the comparisons are made between the haplotypes. The letter behind each taxon indicates the capture locality. For abbreviations see Figure [1](#F1){ref-type="fig"}.](1471-2148-2-4-2){#F2}

![Phylogram based on 357 bp sequences of the cytochrome b gene. Bootstrap method with neighbor joining search and Kimura two parameter distance correction obtained with 10000 replications are used. The tree is rooted using an *Eulemur macaco flavifrons* (EMF) sequence and the comparisons are made between the haplotypes. The letter behind each taxon indicates the capture locality. For abbreviations see Figure [1](#F1){ref-type="fig"}.](1471-2148-2-4-3){#F3}

![Phylogenetic tree based on a 438 bp 12S mitochondrial DNA sequences. Bootstrap method with neighbor joining search and Kimura two parameter distance correction obtained with 10000 replications are used. Only nodes with a bootstrap value greater than 50% are indicated. The tree is rooted using a *Lemur catta* (LCA) sequence (EMBL, accession n°A Y012130) The letter behind each taxon indicates the capture locality. For abbreviations see Figure [1](#F1){ref-type="fig"}.](1471-2148-2-4-4){#F4}

![Phylogram based on a 438 bp 12S mitochondrial DNA sequences. Bootstrap method with neighbor joining search and Kimura two parameter distance correction obtained with 10000 replications are used. The tree is rooted using a *Lemur catta* (LCA) sequence (EMBL, accession n°A Y012130) The letter behind each taxon indicates the capture locality. For abbreviations see Figure [1](#F1){ref-type="fig"}.](1471-2148-2-4-5){#F5}

::: {#T2 .table-wrap}
::: {.caption}
######

Haplotypes and GenBank accession numbers of Hapalemur cytochrome b sequences.
:::

  Species and subspecies/number of individuals   Haplotype/number of individuals   GenBank accession numbers
  ---------------------------------------------- --------------------------------- ---------------------------
  *Hapalemur simus* (HSI)/14                     01/3                              AJ428977
                                                 02/1                              AJ428978
                                                 03/4                              AJ428979
                                                 04/6                              AJ428980
  *Hapalemur aureus* (HAU) / 5                   01/4                              AJ428957
                                                 02/1                              AJ428958
  *Hapalemur griseus meridionalis* (HGM)/16      01/1                              AJ428959
                                                 02/1                              AJ428960
                                                 03/1                              AJ428961
                                                 04/1                              AJ428962
                                                 05/1                              AJ428963
                                                 06/1                              AJ428964
                                                 07/1                              AJ428965
                                                 08/7                              AJ428966
                                                 09/1                              AJ428967
                                                 10/1                              AJ428968
  *Hapalemur griseus ssp* (HGssp) / 50           01/7                              AJ429054
                                                 02/12                             AJ429055
                                                 03/3                              AJ429056
                                                 04/2                              AJ429057
                                                 05/6                              AJ429058
                                                 06/1                              AJ429059
                                                 07/6                              AJ429060
                                                 08/5                              AJ429061
                                                 09/1                              AJ429062
                                                 10/2                              AJ429063
                                                 11/2                              AJ429064
                                                 12/1                              AJ429065
                                                 13/1                              AJ429066
                                                 14/1                              AJ429067
  *Hapalemur griseus alaotrensis* (HGA) / 3      01/1                              AJ428969
                                                 02/2                              AJ428970
  *Hapalemur griseus griseus* HGG)/18            01/1                              AJ428971
                                                 02/12                             AJ428972
                                                 03/1                              AJ428973
                                                 04/1                              AJ428974
                                                 05/1                              AJ428975
                                                 06/2                              AJ428976
  *Hapalemur griseus occidentalis* (HGO) / 9     01/2                              AJ428982
                                                 02/2                              AJ428983
                                                 03/1                              AJ428984
                                                 04/1                              AJ428985
                                                 05/2                              AJ428986
                                                 06/1                              AJ428987
  *Eulemur macaco flavifrons* (EMF)/11           01/11                             AJ428981
:::

The use of the Kimura two parameter method (Table [1](#T1){ref-type="table"}) shows inside S1 a very close distance between HGA and HGG (ranging from 0.00281 to 0.00847), while a larger distance occurs between HGG and HGssp~a~ (0.00847 to 0.02875) as well as between HGG and HGO (0.01412 to 0.03180). In S2 (Table [1](#T1){ref-type="table"}), short distances are observed between HGM and HGssp~b~ (0--0.02855), (in this subclade, the sequence of HGM 02 and HGssp 01 are identical). Larger distances ranging from 0.10473 (HGssp~a~/HGM) to 0.06240 (HGM-HGssp~b~/HGG-HGssp~a~) separates S1 from S2 (Table [1](#T1){ref-type="table"}).

In order to confirm the position of *H. griseus ssp* inside the *H. griseus* subclades, a portion of 12S mitochondrial DNA was amplified and sequenced. The resulted 438 bp fragments were aligned, and the sequences were analyzed with the program PAUP. The trees obtained with HGO, HGG, HGssp and HGM 12S sequences (Figs [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}) also demonstrated a separation of HGssp into two subclades. The first (HGssp~a~), which is composed of animals clustering with HGO and HGG when cytochrome b sequences are analyzed (Figs [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}), also clusters with HGO and HGG when 12S sequences are employed (Figs [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}). The second (HGssp~b~), constructed with animals clustering with HGM when cytochrome b sequences are analyzed (Figs [2](#F2){ref-type="fig"}, [3](#F3){ref-type="fig"}), also clusters with HGM when 12S sequences are employed (Figs [4](#F4){ref-type="fig"}, [5](#F5){ref-type="fig"}).

Discussion
==========

During the last decade, comparisons of mitochondrial DNA sequences have been very useful for the analyses of the phylogenetic species relationships including lemurs \[[@B11]-[@B15],[@B20]\]. In our study, cytochrome b and 12S mtDNA sequences were used, in order to clarify the position of each species and subspecies in the genus *Hapalemur.* Despite the fact that we analysed only short sequences, the genetic distances are considered to be relevant because of the large number of animals involved, since large sample size reduces errors in the estimation of evolutionary relatedness \[[@B11],[@B21]\].

Comparisons of cytochrome b sequences demonstrated that pairwise genetic distances between *H. simus* and *H. aureus* and the other *Hapalemur* (distances ranging from 0.09098 to 0.15338) are the highest. The corresponding phylogram also shows that these taxa are well separated. Our molecular data thus strongly support the species status for *H. simus, H. aureus* and *H. griseus.*

Among the *H. griseus,* the genetic divergence observed between *H. g. meridionalis* and the HGG, HGA, HGssp~a~ and HGO (ranging from 0.06240 to 0.10473) is markedly higher than all other values of the same level of intra *H. griseus* comparison (ranging from 0.00281 to 0.03781). The phylogram also clearly separates the *H. g. meridionalis* from other *H. griseus.* This high genetic divergence in the ranging of those observed between species of lemurs, would support the classification *of H. g. meridionalis* in a separate species *\"H. meridionalis\"* despite the relatively small chromosomal differences observed \[[@B9]\].

Lower distances (ranging from 0.01133 to 0.03781) are observed between *H. griseus* and *H. g. occidentalis.* Moreover, these two forms cluster in the same clade. So, despite the cytogenetic differences existing between these two forms, which differ by two chromosomal rearrangements \[[@B10]\], we propose the maintenance of the subspecies status for *H. g. occidentalis.*

On the basis of our analyses, *H. g. alaotrensis* is indistinguishable from *H. g. griseus.* The very short genetic distances found between *H. g. griseus* and *H. g. alaotrensis* (0.00281 to 0.00847) suggests a combination of these two groups into a single subspecies. However, the differences of the body sizes \[[@B4]\] and the differences in the content of heterochromatin found in both karyotypes \[[@B22]\] support the separation *of H. g. griseus* and *H. g. alaotrensis* in separate subspecies \[[@B23]\], but in no case in species apart as it has been previously proposed \[[@B24]\].

The systematic position of HGssp is more difficult to establish when cytogenetic and molecular data are compared. Cytogenetic data demonstrated the existence of an unique chromosomal polymorphic subspecies characterized by two karyotypes, 2N = 56 and 2N = 54, and their hybrids 2N = 55 \[[@B8]\]. In the view of molecular data, HGssp contains two groups, HGssp~a~ and HGssp~b~, separated by an important genetic distance (0.06240--0.09719) in the range of those observed between species. Each of these two groups contains both karyotypes, 2N = 56 and 2N = 54, as well as their hybrids. The HGssp~a~ are separated from HGG, HGA and HGO by a genetic distance in the range of those observed between subspecies (0.00563--0.03781), while HGssp~b~ appears similar to HGM. Moreover, the HGssp~b~ haplotypes are mixed with those of HGM. Taking into account these results, HGssp~a~ should be considered as belonging to the group of HGG, HGA and HGO, and HGssp~b~ as belonging to the group of HGM.

The existence of two well-separated clades among the *Hapalemur griseus ssp* originating from the Ranomafana region could possibly resulted from either recent mitochondrial DNA introgression or ancestral polymorphism. As the boundaries between HGM and HGssp are still unknown, we could hypothesize that the limits could be close to Ranomafana, allowing hybridization between these two forms. A transfer of mitochrondrial DNA from HGM into the HGssp population could have occurred through a matrilinear process resulting in the HGssp~b~ haplotype. New investigations in areas located in the south of Ranomafana should thus allow the finding of HGM populations containing introgressed HGssp mitochondrial DNAs, unless this transfer occurs only from HGM to HGssp. A second hypothesis is that an ancestral polymorphic population containing both HGM and HGssp haplotypes and a chromosomal polymorphism have diverged in two separated populations. In the population of Ranomafana, the HGM and HGssp haplotypes as well as the chromosomal polymorphism were maintained. In the second population, only the HGM haplotype remained present, and a gain of a large block of heterochromatin gave rise to the karyotype characteristic of the HGM \[[@B9]\].

The comparisons of the phylogenetic trees based on mitochondrial DNA sequences with those previously obtained from cytogenetic data are only partially concordant. In both trees, *H. simus* emerges first, followed by *H. aureus* and then by the different *H. griseus* forms. The cytogenetic data allow to propose an evolutionary tree in which *H. g. occidentalis* emerges first, followed by *H. g. griseus,* and *H. g. meridionalis*\[[@B10]\], whereas on the tree based on cytochrome b and 12S sequences, *H. g. meridionalis* appears as a sister clade of the other *H. griseus.* This difference may be related to the short distances observed inside each clade which allowed no branching order.

Conclusions
===========

Our molecular studies of the *Hapalemur* genus raise the question of the classification of *H. g. meridionalis* in the species status *H. meridionalis.* They also confirm the subspecies status *of H. g. occidentalis* and the absence of arguments in favour of the classification of *H. g. alaotrensis* as a separate species. The sequencing of the *Hapalemur griseus ssp* originating from Ranomafana reveals animals clustering either with the *H. meridionalis* or with the group of *Hapalemur griseus griseus.* No monophyletic clade could be determined in this new cytotype, so that the taxonomic status of the *Hapalemur griseus ssp* remains undefined. As our molecular data did not match the branching sequence within the *H. griseus* group based on cytogenetic data, further investigations including nuclear DNA will be necessary in order to resolve this issue.

Materials and methods
=====================

Animal studied
--------------

Six survey were organised from 1997 to 2001 allowing the capture of 115 animals in bamboo forests extending from the north to the south of Madagascar. Animals were captured using blowpipe projections and then sexed, weighted and measured. Skin samples were cut off under general anaesthesia with a 2 mg/kg injection of ketamine solution (Ketalar^®^ Parke-Davis) and conserved deep frozen in liquid nitrogen. The different capture areas and the number of animals are indicated in Figure [1](#F1){ref-type="fig"}. As the *H. griseus* taxonomy is essentially based on cytogenetic criteria, karyotypes were made in order to confirm the species and subspecies rank. From the north to the south, the following subspecies were caught: a total of nine *H. g. occidentalis* (four at Analamera (a), two at Ambato (b), two at Ambakoany (c), and one at Maroantsetra (d)). Three *H. g. alaotrensis* at the Alaotra lake (e); 18 *H. g. griseus* at Maromiza (f); 50 *H. griseus ssp.* (37 at Ranomafana (h), five at Ambolomavo (i), eight at Kianjavato (j)); 16 *H. g. meridionalis* (14 in Andohahela (k) and two in Mandena (l)). In addition, 12 *H. simus* (four from Ranomafana (h), and eight from Ambolomavo (i)), as well as two *H. simus* and five *H. aureus* from the Zoological Park of Tsimbazaza (g) were studied. Animals were released at their capture location, immediately after recovery from anaesthesia, except two *H. g. occidentalis* captured in Ambato as well as three *H. g. alaotrensis* which are kept in the Zoological Park of Mulhouse and one *H. g. occidentalis* from Maroan setra which is kept in the private Zoological Park of Mandraka. As outgroup, we used a sample *of Eulemur macaco flavifrons,* from one of the individuals captured in the Sahamalaza forest for an earlier study \[[@B25]\].

DNA extraction
--------------

DNA samples were extracted from the skin biopsies using the standard proteinase K digestion followed by a phenol chloroform extraction as described by Sambrook *et al.*\[[@B26]\] with minor modifications. Small pieces of skin (\~9mm^2^) were suspended in 200μl of extraction buffer (Tris 0.2M pH 8.4; KCl 0.5M; proteinase K: 5 mg/ml), and incubated at 37°C overnight. The samples were than heated at 95°C and mixed with an equal volume (200μl) of phenol/chloroform (1/1). After centrifugation (6 mn at 8500 g) the aqueous part was precipitated with 2 volumes of absolute ethanol at -20°C in presence of 1/10 (V/V) of ammonium acetate 5M. After centrifugation (5 mn at 8500 g), pellets were rinsed with 200μl of 70% ethanol and dried at 37°C. Pellets were resuspended in sterile double-distilled water and the concentration of the DNAs were measured by absorption at 260 nm. DNA samples were then stored at -30°C.

Amplification conditions
------------------------

### Cytochrome b

The polymerase chain reaction (PCR) was employed to generate a double-stranded fragment of 357 bp corresponding to a part of the mitochondrial cytochrome b gene. Each amplification was performed in the presence of 20 mM Tris-HCl pH 8.4, 50 mM KCl, 1.5mM MgCl~2~ (Gibco BRL), 200 μg BSA, 0.5 μM of each primer, 5U of Taq DNA polymerase (Perkin Elmer Cetus), 200 μM of each dNTP (Boehringer Mannheim) and 360ng of template DNA in a volume of 200 μl. The following primers derived from those described by Kocher *et al.*\[[@B27]\] were employed: Pr181 (5\'-CCATCCAACATGTCAGCATGATGAAA-3\') and Pr182 (5\'-CCCTCAGAATGATATTTGTCCTCA-3\'). Reactions were done in a Perkin Elmer Cetus DNA thermocycler 480 as follows: predenaturation (7 mn at 93 °C) and 35 cycles of denaturation (30 s at 94°C), annealing (45 s at 42°C), extension (1 mn 30 s at 72°C), followed by a final extension step (10 mn at 72°C).

### 12S mitochondrial DNA

The same PCR conditions than those described for the cytochrome b amplifications were applied, with a concentration of 2 mM MgCl~2~ and the following set of primers: Pr179 (5\'-AAACTAGGATTAGATACCCTATTAT-3\') and Pr180 (5\'-AAGAGCGACGGGCGATGTGT-3\'). Amplifications were performed in a Perkin Elmer Cetus DNA thermocycler 480 as follows: predenaturation (10 mn at 94°C) and 40 cycles of denaturation (30 s at 94°C), annealing (45 s at 45°C), extension (1 mn 30 s at 72°C), followed by a final extension step (10 mn at 72°C).

### Sequences

Amplification products were electrophoresed on 1.3% agarose gels in TBE buffer (Tris base 87 mM, boric acid 89 mM, EDTA 2 mM, pH 8.0) in the presence of 0.5 μg/ml of ethidium bromide. Electrophoresis were performed at 200 mA for 2 h in TBE. Gels were than examined and photographed under UV light with a Polaroid system. The major bands on the gels were cut and centrifuged (8500 g 10 mn) in order to recover the DNA fragments \[[@B28]\]. The fragments were then precipitated with 0.1 volumes of ammonium acetate 5 M and 2 volumes of absolute ethanol at -20°C. After centrifugation (8500 g 10-mn), the pellets were air dried, resuspended in 50 μl of sterile bidistilled water and sequenced on an automatic ABI PRISM sequencer with the Taq dye deoxy terminator cycle sequencing kit. Each sample was sequenced from 5\' to 3\' and 3\' to 5\'.

Cytochrome b sequences were aligned and for each taxa, similar sequences were grouped under one haplotype. These haplotypes and the EMBL GenBank accession numbers are listed in table [2](#T2){ref-type="table"}. EMBL GenBank accession numbers for the 12S sequences are listed in table [3](#T3){ref-type="table"}.

::: {#T3 .table-wrap}
::: {.caption}
######

GenBank accession numbers of Hapalemur 12S sequences.
:::

  Species/subspecies                       N°          GenBank accession numbers
  ---------------------------------------- ----------- ---------------------------
  *Hapalemur griseus meridionalis* (HGM)   25          AJ429205
                                           29^\*^      AJ429206
  *Hapalemur griseus ssp* (HGssp)          41          AJ429207
                                           43          AJ429208
                                           59          AJ429209
  *Hapalemur griseus griseus* (HGG)        06          AJ429210
                                           10          AJ429211
  *Hapalemur griseus occidentalis* (HGO)   19 ^\*\*^   AJ429212
                                           20          AJ429213
                                           23          AJ429214
                                           24          AJ429215

^\*^ 12S sequence of HGM29 is similar to HGssp40, HGssp42 and HGssp73. ^\*\*^ 12S sequence of HGO19 is similar to HGO22.
:::

The aligned sequences were analysed using neighbor-joining and maximum parsimony methods with the computer program Pylogenetic Analysis Using Parsimony (PAUP) 4.0b.4a \[[@B19]\]. Genetic distances (d) measured in neighbor-joining analyses were calculated using the Kimura two parameter method \[[@B18]\] with the following formula: d=-1/21n \[(1-2P-Q)Xsqr(1-2Q)\], with P=transitions/positions scored and Q=transversions/positions scored. For neighbor-joining and maximum parsimony analyses, bootstraps of 10000 replicates were performed to examine the relative support of each relationship in the resultant topologies. Maximum parsimony trees were calculated via fast stepwise addition with random addition sequence.

Acknowledgements
================

This work was partially supported by the Association Européenne pour l\'Etude et la Conservation des Lémuriens (A.E.E.C.L.), the DREIF-MENESR (EA3428), and the Institut de Génétique et de Biologie Moléculaire et Cellulaire (Pr. P. Chambon). The authors thank the Commission Tripartite of the Malagasy Government and the Ministère pour la Production Animale et des Eaux et Forêts for their permission to capture the animals, to take samples and for delivery CITES. Thanks are also due to Dr J. Ganzhorn for the correction of the manuscript, Mrs M. Lavaux for her secretarial assistance, Dr B. Ravaoarimanana and M. Hauwy for their helpful comments, and to S. Vicaire (IGBMC, Strasbourg) for sequencing the cytochrome b and 12S fragments. Special thanks for insight comments on the cladograms are due to Dr. D. Montagnon.


Background
==========

Fruit-set and growth of several horticultural plants are negatively affected by adverse environmental conditions. In general, sub and/or supra-optimal temperatures negatively affect reproductive processes and therefore curtail fruit production \[[@B1],[@B2]\]. Under greenhouse cultivation, low temperature, insufficient light intensity, and/or high humidity drastically reduce fruit productivity and quality in eggplant and other species. Moreover, environmental conditions often met in open field cultivation such as drought and high temperatures have a negative effect on fruit productivity and quality in eggplant and other species (e.g. tomato).

Parthenocarpic fruit development (i.e. fruit-set and growth without fertilization) can significantly aid in the resolution of the aforementioned problems. Parthenocarpy can be triggered by exogenous factors, such as plant growth regulators, or it can be achieved by genetic factors. Genes causing parthenocarpic development have been identified in several plant species \[[@B3]-[@B5]\], and parthenocarpic eggplant varieties (e.g. Talina, Galine) have been introduced in the production process (e.g. protected cultivation). However, during winter cultivation of eggplant varieties in unheated greenhouses in the Mediterranean area, the negative effect of suboptimal environmental conditions on fruit production is usually counteracted by treating flower buds with plant growth regulators. Phytohormonal treatments make the production process more expensive due to the cost of both chemicals and labor.

In principle, the genetic engineering of plants allows one to confer a trait of interest to different species and within a species to all the varieties of interest. To confer parthenocarpic fruit development, a chimeric gene has been constructed \[[@B6]\]. Specifically, the *DefH9-iaaM* gene contains the coding region of the *iaaM* gene from *Pseudomonas syringae* pv. *savastanoi* under the control of the placenta and ovule-specific promoter from the *DefH9* gene from *Anthirrinum majus*\[[@B6]\]. The *iaaM* gene codes for a tryptophan monoxygenase, which produces indolacetamide that in turn, is either chemically or enzymatically converted to the auxin indole-3-acetic acid. To date, the *DefH9-iaaM* chimeric gene has been shown to cause parthenocarpic fruit development in tobacco \[[@B6]\], eggplant \[[@B6]\], tomato \[[@B7],[@B8]\], strawberry and raspberry \[[@B9]\].

We have previously shown that *DefH9-iaaM* eggplants outperform control plants during protected winter cultivation by an average of 33% \[[@B10]\]. The present manuscript presents data on the agronomic performance of *DefH9-iaaM* eggplant hybrids during spring and summer cultivation. Seed-derived F1 plants perfectly reflects the real agronomical situation of eggplant production. In addition, they have allowed to demonstrate that the transgene is active after meiosis and give satisfactory results in the hemizygous state. Different types of eggplant hybrids have been evaluated during springtime in unheated greenhouses in two different locations. To evaluate GM parthenocarpic eggplants under optimal environmental conditions, a single trial has been carried out using two different genotypes under standard open field cultivation during summertime. Furthermore, we demonstrate that the *DefH9-iaaM* gene is expressed during late stages of fruit development.

Results and discussion
======================

Greenhouse spring production
----------------------------

Spring production was evaluated in trials performed in two different locations: Monsampolo and Pontecagnano. Spring production was divided into early production, consisting of the first four harvests, and total production, consisting of sixteen harvests. Early spring production corresponds to the cultivation period from March to the first half of May, with temperatures somewhat low for fruit-set and growth. During this period, the average minimum and maximum temperatures ranged from 7° to 17°C in southern Italy (Pontecagnano) and from 14° to 17°C in central Italy (Monsampolo). The transgenic parthenocarpic hybrids gave a significantly higher yield, on the average a six-fold increase during early production, in comparison to their controls (Table [1](#T1){ref-type="table"}). The increment in the number of fruits per plant and the higher average weight of the fruits were the main causes of the increased early spring production of the parthenocarpic hybrids. The suboptimal/adverse environmental conditions did not affect the growth of GM parthenocarpic fruits and the average fruit weight was significantly higher in the GM eggplants in comparison to untransformed controls. The traditional parthenocarpic cultivar Talina produced fruits with an average weight similar to those of GM eggplant fruits (Table [1](#T1){ref-type="table"}). However, due to the higher number of fruits per plant, the productivity of the transgenic parthenocarpic hybrids was also increased, by an average of five-fold, when compared to cv Talina (Table [1](#T1){ref-type="table"}). During the whole harvesting period, the GM fruits were always seedless (Fig. [1](#F1){ref-type="fig"}) and were normal in both size and shape.

![Eggplant fruits of the transgenic parthenocarpic hybrid P1 from the greenhouse spring trial. Left: fruit at the stage of commercial ripeness; middle: an overripe eggplant fruit from a border plant; right: a longitudinally cut fruit showing the complete absence of seeds.](1472-6750-2-4-1){#F1}

::: {#T1 .table-wrap}
::: {.caption}
######

Eggplant production of parthenocarpic hybrids and their respective controls at springtime.
:::

  Genotype   Early production   Total production
  ---------- ------------------ ------------------ --------- -------- ------- ---------
  P1         488 b              1.9 a              268.3 a   2241 a   8.4 a   253.9 a
  P2         695 a              2.6 a              290.8 a   2288 a   8.6 a   260.2 a
  C1         75 c               0.4 b              227.0 b   1547 b   7.8 a   187.5 b
  P5         522 b              2.0 a              281.8 a   2163 a   9.2 a   230.5 a
  C2         116c               0.7 b              170.6 b   1574 b   9.3 a   167.3 b
  Talina     114c               0.3 b              270.5 a   2360 a   9.4 a   229.9 a

For each trait at least one common letter indicates no significant difference according to the Duncan test (P = 0.05). Mean values of yields per plant, number of fruits per plant, and fruit weight of three transgenic parthenocarpic hybrids (P1, P2 and P5), two controls (C1 and C2) and the commercial cultivar Talina. C1 hybrid plants represent the controls of P1 and P2 transgenic hybrid plants. C2 hybrid plants are the controls of the P5 transgenic hybrids. Data are the average of the Monsampolo and Pontecagnano locations. The experiments were carried out in greenhouse at springtime.
:::

The increased productivity of GM hybrids characterised both the early spring production (i.e. the first four harvests) and the whole spring production cycle (i.e. sixteen harvests). During the whole spring production cycle, the hybrids P1 and P2 gave an average yield that was 46% higher with respect to the corresponding control C1 (Table [1](#T1){ref-type="table"}). The hybrid P5 gave a 37% higher yield with respect to its control C2. The average total number of fruits produced per plant in both locations was similar in all the hybrids (8--9 fruits/plant). However, the higher average weight of the GM fruits led to a higher total yield of transgenic hybrids with respect to their controls. When considering the whole spring cultivation cycle, the parthenocarpic cultivar Talina gave a total production that was not significantly different from either of the three GM hybrids (Table [1](#T1){ref-type="table"}).

Open field (summer) production
------------------------------

Summer production was evaluated in an open field trial carried out during the optimal period of eggplant cultivation. Plants were transplanted on May 20^th^ and the last harvest took place on September 11^th^. The early production of the transgenic hybrids, consisting of the first three harvests, was significantly higher than that of the untransformed hybrids (Table [2](#T2){ref-type="table"}): P1 and P10 hybrids yielded, respectively, 36% and 76% more than their corresponding controls, C1 and C10. The difference in productivity between P1 and C1 hybrids, which have long-shaped fruits, was caused by the higher average weight of GM fruits. When comparing P10 and C10 hybrids, which have sub-oval fruits, the higher yield obtained with GM plants during the early harvesting period was due to the increased number of fruits per plant.

::: {#T2 .table-wrap}
::: {.caption}
######

Eggplant production of parthenocarpic hybrids and their respective controls at summertime.
:::

  Genotypes   Early production   Total production
  ----------- ------------------ ------------------ ------- --------- -------- -------
  P1          1158a              2.7 a              433 b   3039 a    9.1 a    335 b
  C1          846 b              2.4 a              344 c   2211 b    7.7 ab   288 b
  P10         1287 a             2.4 a              553 a   2791 ab   6.8 bc   410 a
  C10         731 b              1.3 b              541 a   2386 ab   5.7 c    415 a

For each trait, at least one common letter indicates no significant difference according to the Duncan test (P = 0.05). Mean value of yields per plant, number of fruits and fruit weight of two transgenic hybrids (P1 and P10) and their corresponding untransformed hybrids (C1 and C10), cultivated during summertime under open field conditions.
:::

Consisting of ten harvests, the total production of P1 hybrids was 37% higher than control C1 eggplants (Table [2](#T2){ref-type="table"}). The difference in total yield between P1 and control C1 hybrids was statistically significant and due both to the higher number of fruits/plant and to the increased weight of GM fruits. It is noteworthy to point out that when considered individually, neither trait (number of fruits/plant or fruit weight) showed statistically significant differences between the GM and untransformed plants (Table [2](#T2){ref-type="table"}). Although higher in P10 hybrids in comparison to its control C10, the total yield (the number and average weight of the fruits) was not significantly different between the two. During the whole harvesting period, fruits from both P1 and P10 parthenocarpic hybrids were always seedless (Fig. [2](#F2){ref-type="fig"}), whilst control fruits always had seeds. Therefore, under open field cultivation, the *DefH9-iaaM* gene had a positive influence on fruit quality, as GM *DefH9-iaaM* fruits were always seedless. Fruit quality affects the economic value of eggplant production.

![Eggplant fruits from the open field summer trial. Left, uncut and cut fruit of the transgenic hybrid P10; Right, cut and uncut fruit of the C10 control hybrid.](1472-6750-2-4-2){#F2}

Although the environmental conditions were optimal and consequently did not affect negatively fruit-set, the *DefH9-iaaM* gene caused both faster development and growth of the fruits as indicated by the increased early-summer production (the first three harvests). In this regard, it is worthwhile to stress that expression of the *DefH9-iaaM* gene takes place in the placenta and ovules before pollen development. As a consequence, in GM parthenocarpic plants fruits are seedless and fruit development initiates well before non-GM controls \[[@B11]\].

In all trials we have never used homozygous lines because growers mostly employ F1 hybrids. The use of hemizygous primary transformants as pollinator plants allowed us to obtain in rather short time, by *in vivo* selection for kanamycin resistance, F1 plants transgenic for the *DefH9-iaaM* gene. Young, healthy and vigorously growing plants did not produce seeds. However, it is possible to obtain seeds from aged *DefH9-iaaM* transgenic plants both by selfing and crossing. By exploiting the delayed female fertility we have produced the homozygous plants needed as male parents for rapid seed multiplication of F1 eggplant hybrids. Therefore, the female sterility of young and mature plants is not an insuperable hindrance for mass propagation and commercial fruit production.

Expression of the *DefH9-iaaM* gene takes place during both flower and fruit development in transgenic parthenocarpic eggplants
-------------------------------------------------------------------------------------------------------------------------------

The *DefH9* gene is expressed specifically in the placenta and ovules during early phases of flower development \[[@B6]\]. To determine whether expression of the *DefH9-iaaM* gene also occurs during later stages of fruit growth and whether it is influenced by pollen fertilization, mRNA from transgenic flowers and fruits obtained either from emasculated or hand pollinated flowers was analyzed by RT-PCR at different stages of development, until the fruit attained a length of 28 cm. An amplicon of 161 bp corresponding to the spliced *DefH9-iaaM* mRNA was detected in all stages analyzed (Fig. [3](#F3){ref-type="fig"}, lanes 2--6). Thus, the presence of the mRNA of the *DefH9-iaaM* gene and consequently its action is most likely not limited to early stages of flower and fruit development. Pollination did not affect the steady state level of *DefH9-iaaM* mRNA (Fig. [3](#F3){ref-type="fig"}, compare lane 5 and lane 6).

![Expression analysis of the *DefH9-iaaM* gene by competitive RT-PCR during flower and fruit development. Untransformed plant (lane 1); 5, 8, 12 mm long flower buds (lanes 2, 3, 4, respectively); 40 mm long hand-pollinated fruit (lane 5); 280 mm long emasculated fruit (lane 6). An internal standard of 351 bp is present in all lanes.](1472-6750-2-4-3){#F3}

Treatment with auxin often causes parthenocarpic development in several plant species \[for review, see: \[[@B12]\]\]. However, in some species and/or varieties, to efficiently sustain fruit growth, the hormonal treatment of the flowers must be repeated \[[@B13]\]. The finding that *DefH9-iaaM* mRNA is also present during later stages of fruit development is consistent with the interpretation that in *DefH9-iaaM* parthenocarpic plants, the placenta, the ovules, and the tissues derived therefrom are a source of auxin during the whole growth of the fruit. As a consequence, they efficiently sustain fruit growth.

Conclusions
===========

The data hereby presented show the positive influence of the *DefH9-iaaM* parthenocarpic gene on eggplant productivity under both greenhouse (spring) and open field (summer) cultivation. Taking into account the data previously obtained under winter greenhouse cultivation \[[@B10]\], we conservatively estimate that the overall increase in productivity is at least 30--35%. The increase in productivity of *DefH9-iaaM* eggplants is mainly due to a drastically improved fruit-set under sub-optimal temperatures and to an enhanced fruit growth and weight. Fruit quality is also improved because the fruits are seedless and do not show a placental cavity. The qualitative improvement of *DefH9-iaaM* eggplant fruits is interesting both for the fresh market and for the processing industry. During early spring greenhouse production, *DefH9-iaaM* parthenocarpic hybrids always gave fruits with an average weight suitable for fresh market commercialization, while untransformed hybrids, under sub-optimal conditions, rarely produced commercial fruits. Thus, the *DefH9-iaaM* gene quantitatively and qualitatively improves eggplant production under both greenhouse and open field cultivation. In all genotypes tested the *DefH9-iaaM* gene had a very positive effect on production and quality parameters. Such findings are of paramount importance as the hybrids tested have the same genetic background that the relative controls, except for the presence of the *DefH9-iaaM* gene. The *DefH9-iaaM* gene, already known to be expressed in the placenta and ovules during early phases of flower development, is expressed also in mature fruits, most likely in tissues derived from the ovules.

From an economical standpoint, the main advantages conferred to eggplant by the *DefH9-iaaM* gene are: i) production of marketable fruits under environmental conditions adverse for fruit-set and growth; ii) reduction of cultivation costs (energy, phytohormones and labor) necessary for off-season and open field eggplant cultivation; and iii) enhancement of fruit quality. Last but not least, contrary to conventional wisdom, these advantages have been achieved without the use of either male or female sterility genes.

Materials and methods
=====================

Greenhouse spring cultivation
-----------------------------

Trials were carried out in central Italy (Monsampolo del Tronto-AP) and in southern Italy (Pontecagnano-SA) (approval of the Italian Ministry of Health N° B/IT/97-29). The greenhouses were rather similar and made of galvanized steel and covered with plastic polyethylene (0.12 mm thick). An apparatus for drip-irrigation was used and the soil was completely mulched. A complete randomized block design with three replicate hybrid genotypes was adopted. Each experimental plot measured 3.12 m^2^ and contained eight plants in a double row. Transplanting was performed on March 3^rd^ in southern Italy and on March 27^th^ in central Italy. The P1, P2, P5, C1, C2 and the commercial Talina hybrids were employed. Transgenic parthenocarpic hybrids P1, P2 and P5 were obtained by crossing (as male parent) the primary transgenic plant DR2 *iaaM* \#34-1 with the line Tal 1/1 (P1), the primary transgenic plant DR2 *iaaM* \#28-1 with Tal 1/1 (P2) and the transgenic plant Tal 1/1 *iaaM* \#1-1 with the line Tina (P5). The hybrids P1 and P2 are homologous to C1 (DR2 × Tal1/1), except for the presence of the *DefH9-iaaM* gene integrated in their genome. The transgenic hybrid P5 is homologous to its untransformed control C2 (Tal1/1 × Tina). DR2 and Tina are parental lines obtained through classical breeding, Tal1/1 is a double haploid line derived from anther culture of the F1 commercial cultivar Talina. The segregation of the marker gene *nptII* was checked by spraying the plants with kanamycin \[[@B14]\] and allowed for the conclusion that the transgenes segregate as a single locus in the backcrossed progenies of the three independent events analyzed (Tal *iaaM* 1-1: χ^2^ = 0.01065, P = 0.917; DR2 *iaaM* 34-1: χ^2^ = 0.0496 P = 0.824; DR2 *iaaM* 28-1 χ^2^ = 0.06467 P = 0.799). Southern blot analysis showed that DR2 *iaaM* 28-1 and 34-1 had a single copy of the transgene, while Tal *iaaM* 1-1 had three copies of the transgene (Fig [4](#F4){ref-type="fig"}). Since the interaction genotype/location was not significant for the yield, the data were computed as average of the two locations and subjected to analysis of variance according to a randomized complete block design. Duncan\'s Multiple Range Test (P = 0.05) was used for means separations.

![Southern blot analysis of transgenic eggplants. Numbers above the lanes indicate the independent transgenic plant DR2iaaM\#28-1 (28), DR2iaaM\#34-1 (34) and *Tal1/1iaaM\#1-1* (Tal1/1-1). Cont indicates untransformed plants, i.e. DR2 and Tal1/1, respectively. The probe used corresponds to the *DefH9* regulatory region.](1472-6750-2-4-4){#F4}

Open field (summer) cultivation
-------------------------------

The open field trial was carried out under open field conditions at Monsampolo del Tronto (approval of the Italian Ministry of Health B/IT/99/21). Two transgenic parthenocarpic hybrids were tested: the hybrid P1 (Tal1/1 × DR2 *iaaM* \#34-1) with elongated fruits and the hybrid P10 (UGA × Tal1/1 *iaaM* \#1-1) with sub-oval fruits were compared to their homologous non-transgenic controls C1 (DR2 × Tal1/1) and C10 (UGA × Tal1/1), respectively. The UGA line has oval dark purplish fruits and it has been provided by Dr. S.C. Phatak. A complete randomized block design with the hybrids replicated four times was adopted. Each experimental plot measured 11.7 m^2^ and contained 30 plants in a double row. Transplanting was performed on May 10^th^.

Early spring production consisted of the first four harvests (i.e. 4 out of 16 harvests during the whole production cycle), while early summer production, whose cultivation cycle consists of ten harvests, corresponds to the first three harvests. For all trials the number and weight of fruits were recorded. In addition, fruit sample for each harvest and replication was cut to check for the presence of seeds. Data were computed for the early harvesting time and for the whole harvesting season. Analysis of variance was performed according to a randomized complete block design. Duncan\'s Multiple Range Test (P = 0.05) was used for means separations.

Plant DNA isolation and Southern blot analysis
----------------------------------------------

High-molecular-weight DNA was isolated from the young leaves of transgenic and untransformed eggplants by using Plant DNAzol (Invitrogen), according to the manufacturer\'s instructions. Ten μg of DNA was digested overnight with 80 units of *KpnI* (Promega) in a volume of 500 μl, separated on a 0.7% agarose gel and transferred to Hybond N (Amersham Pharmacia Biotech). A 1350 bp fragment of the *DefH9* gene was used as a radiolabeled probe. The membrane was hybridized overnight in 5X SSC/50% formamide (Sigma) at 42°C and washed two times for 15 min. in 2 × SSC/0.1% SDS, and two times for 15 min. in 0.1 × SSC/0.1% SDS at 42°C. Signals were detected using Kodak X-OMAT AR5 film (Sigma).

RT-PCR analysis
---------------

Semiquantitative (competitive) PCR analysis was carried out for 38 cycles (annealing temperature 63°C) using as template cDNA (8 ng) obtained by priming poly(A)^+^ mRNA with an *iaaM* specific primer (5\'-AATAGCTGCCTATGCCTCCCGTCAT-3\'). The mRNA was extracted from either young flower buds (5,8,11 mm) or eggplant fruits (placental tissue from fruits either 40 or 280 mm long). As an internal standard, 0.5 fg of a 600 bp long *DefH9* cDNA fragment was used in the PCR assay. To amplify the 161 bp long amplicon an *iaaM* specific primer (5\'-GGGTGAATTAAAATGGTCATACAT-3\') and a *DefH9* specific primer (5\'-CTTTGGAACTCGTGTTGAGCTCTCA-3\') were used. For the internal standard, a 3\' primer (5\'-TGAGCATTGATCTCCTGAGTGGTGT-3\') together with the *DefH9* specific primer were used to produce the 351 bp long amplicon. The PCR assays were performed with a thermostable DNA polymerase mixture (Expand High Fidelity PCR system, Roche) in presence of 3 μCi of ^32^P dCTP. The intensity of the bands was quantified by using an Instant Imager (Packard, Meriden, CT).

Acknowledgements
================

This research was partially supported by the Consiglio Nazionale delle Ricerce (Progetto Finalizzato Biotecnologie II) and by the Ministero Politiche Agricole e Forestali Progetto \"Biotecnologie vegetali\". We thank Prof. Phatak, University of Georgia (USA) for the UGA line.