Performance of a Web-Based Clinical Diagnosis Support System for Internists BACKGROUND Clinical decision support systems can improve medical diagnosis and reduce diagnostic errors . Older systems , however , were cumbersome to use and had limited success in identifying the correct diagnosis in complicated cases . INTRODUCTION The best clinicians excel in their ability to discern the correct diagnosis in perplexing cases . This skill requires an extensive knowledge base , keen interviewing and examination skills , and the ability to synthesize coherently all of the available information . Unfortunately , the level of expertise varies among clinicians , and even the most expert can sometimes fail . There is also a growing appreciation that diagnostic errors can be made just as easily in simple cases as in the most complex . Given this dilemma and the fact that diagnostic error rates are not trivial , clinicians are well-advised to explore tools that can help them establish correct diagnoses . Clinical diagnosis support systems -LRB- CDSS -RRB- can direct physicians to the correct diagnosis and have the potential to reduce the rate of diagnostic errors in medicine .1,2 The first-generation computer-based products -LRB- e.g. , QMR -- First Databank , Inc , CA ; Iliad -- University of Utah ; DXplain -- Massachusetts General Hospital , Boston , MA -RRB- used precompiled knowledge bases of syndromes and diseases with their characteristic symptoms , signs , and laboratory findings . The user would enter findings from their own patients selected from a menu of choices , and the programs would use Bayesian logic or pattern-matching algorithms to suggest diagnostic possibilities . Typically , the suggestions were judged to be helpful in clinical settings , even when used by expert clinicians .3 These diagnosis support systems were also useful in teaching clinical reasoning .4,5 Surprisingly and despite their demonstrated utility in experimental settings , none of these earlier systems gained widespread acceptance for clinical use , apparently related to the considerable time needed to input clinical data and their somewhat limited sensitivity and specificity .3,6 A study of Iliad and QMR in an emergency department setting , for example , found that the final impression of the attending physician was found among the suggested diagnoses only 72 % and 52 % of the time , respectively , and data input required 20 to 40 minutes for each case .7 In this study , we evaluated the clinical performance of `` Isabel '' -LRB- Isabel Healthcare Inc , USA -RRB- , a new , second generation , web-based CDSS that accepts either key findings or whole-text entry and uses a novel search strategy to identify candidate diagnoses from the clinical findings . The clinician first enters the key findings from the case using free-text entry -LRB- see Fig 1 -RRB- . There is no limit on the number of terms entered , although excellent results are typically obtained with entering just a few key findings . The program includes a thesaurus that facilitates recognition of terms . The program then uses natural language processing and search algorithms to compare these terms to those used in a selected reference library . For Internal Medicine cases , the library includes 6 key textbooks anchored around the Oxford Textbooks of Medicine , 4th Edition -LRB- 2003 -RRB- and the Oxford Textbook of Geriatric Medicine and 46 major journals in general and subspecialty medicine and toxicology . The search domain and results are filtered to take into account the patient 's age , sex , geographic location , pregnancy status , and other clinical parameters that are either preselected by the clinician or automatically entered if the system is integrated with the clinician 's electronic medical record . The system then displays a total of 30 suggested diagnoses , with 10 diagnoses presented on each web page -LRB- see Fig. 2 -RRB- . The order of listing reflects an indication of the matching between the findings selected and the reference materials searched but is not meant to suggest a ranked order of clinical probabilities . As in the first generation systems , more detailed information on each diagnosis can be obtained by links to authoritative texts . Figure 1The data-entry screen of the Isabel diagnosis support software . The clinician manually enters the age , gender , locality , and specialty . The query terms can be entered manually from the key findings or findings can be pasted from an existing write-up . This patient was ultimately found to have a B-cell lymphoma secreting a cryo-paraproteinFigure 2The first page of results of the Isabel diagnosis support software . Additional diagnoses are presented by selecting the ` more diagnoses ' box The Isabel CDSS was originally developed for use in pediatrics . In an initial evaluation , 13 clinicians -LRB- trainees and staff -RRB- at St Mary 's Hospital , London submitted a total of 99 case scenarios of hypothetical case presentations for different diagnoses , and Isabel displayed the expected diagnosis in 91 % of these cases .8 Out of 100 real case scenarios gathered from 4 major teaching hospitals in the UK , Isabel suggested the correct diagnosis in 83 of 87 cases -LRB- 95 % -RRB- . In a separate evaluation of 24 case scenarios in which the gold standard differential diagnosis was established by two senior academic pediatricians , Isabel decreased the chance of clinicians -LRB- a mix of trainees and staff clinicians -RRB- omitting a key diagnosis by suggesting a highly relevant new diagnosis in 1 of every 8 cases . In this study , the time to enter data and obtain diagnostic suggestions averaged less than 1 minute .9 The Isabel clinical diagnosis support system has now been adapted for adult medicine . The goal of this study was to evaluate the speed and accuracy of this product in suggesting the correct diagnosis in a series of complex cases in Internal Medicine . METHOD We considered 61 consecutive `` Case Records of Massachusetts General Hospital '' -LRB- New England Journal of Medicine , vol . 350:166 -- 176 , 2004 and 353:189 -- 198 , 2005 -RRB- . Each case had an anatomical or final diagnosis , which was considered to be correct by the discussants . We excluded 11 cases -LRB- patients under the age of 10 and cases that focused solely on management issues -RRB- . The 50 remaining case histories were copied and pasted into the Isabel data-entry field . The pasted material typically included the history , physical examination findings , and laboratory test results , but data from tables and figures were not submitted . Beyond entering the patient 's age , sex , and nationality , the investigators did not attempt to otherwise tailor the search strategy . Findings were compared to the recommended -LRB- but slightly more time consuming -RRB- strategy of entering discrete key findings , as compiled by a senior internist -LRB- MLG -RRB- . Because the correct diagnosis is presented at the end of each case , data entry was not blinded . RESULTS Using the recommended method of manually entering key findings , the list of diagnoses suggested by Isabel contained the correct diagnosis in 48 of the 50 cases -LRB- 96 % -RRB- . Typically 3 -- 6 key findings from each case were used . The 2 diagnoses that were not suggested -LRB- progressive multifocal encephalopathy and nephrogenic fibrosing dermopathy -RRB- were not included in the Isabel database at the time of the study ; thus , these 2 cases would never have been suggested , even with different keywords . Using the copy/paste method for entering the whole text , the list of diagnoses suggested by Isabel contained the correct diagnosis in 37 of the 50 cases -LRB- 76 % -RRB- . Isabel presented 10 diagnoses on the first web page and 10 additional diagnoses on subsequent pages up to a total of 30 diagnoses . Because users may tend to disregard suggestions not shown on later web pages , we tracked this parameter for the copy/paste method of data entry : The correct diagnosis was presented on the first page in 19 of the 37 cases -LRB- 51 % -RRB- or first two pages in 28 of the 37 cases -LRB- 77 % -RRB- . Similar data were not collected for manual data entry because the order of presentation depended on which key findings were entered . Both data entry approaches were fast : Manually entering data and obtaining diagnostic suggestions typically required less than 1 minute per case , and the copy/paste method typically required less than 5 seconds . DISCUSSION Diagnostic errors are an underappreciated cause of medical error ,10 and any intervention that has the potential to produce correct and timely medical diagnosis is worthy of serious consideration . Our recent analysis of diagnostic errors in Internal Medicine found that clinicians often stop thinking after arriving at a preliminary diagnosis that explains all the key findings , leading to context errors and ` premature closure ' , where further possibilities are not considered .11 These and other errors contribute to diagnoses that are wrong or delayed , causing substantial harm in the patients affected . Systems that help clinicians explore a more complete range of diagnostic possibilities could conceivably reduce these types of error . Many different CDSSs have been developed over the years , and these typically matched the manually entered features of the case in question to a database of key findings abstracted from experts or the clinical literature . The sensitivity of these systems was in the range of 50 % -- 60 % , and the time needed to access and query the database was often several minutes .3 More recently , the possibility of using Google to search for clinical diagnoses has been suggested . However , a formal evaluation of this approach on a subset of the same `` Case Records '' cases used in our study found a sensitivity of 58 % ,12 in the range of the first-generation CDSSs and unacceptably low for clinical use . The findings of our study indicate that CDSS products have evolved substantially . Using the Isabel CDSS , we found that data entry takes under 1 minute , and the sensitivity in a series of highly complex cases approached 100 % using entry of key findings . Entry of entire case histories using copy/paste functionality allowed even faster data entry but reduced sensitivity . The loss of sensitivity seemed primarily related to negative findings included in the pasted history and physical -LRB- e.g. , `` the patient denies chest pain '' -RRB- , which are treated as positive findings -LRB- chest pain -RRB- by the search algorithm . There are several relevant limitations of this study that make it difficult to predict how Isabel might perform as a diagnostic aid in clinical practice . First , the results obtained here reflect the theoretical upper limit of performance , given that an investigator who was aware of the correct diagnosis entered the key findings . Further , clinicians in real life seldom have the wealth of reliable and organized information that is presented in the Case Records or the time needed to use a CDSS in every case . To the extent that Isabel functions as a ` learned intermediary ' ,13 success in using the program will also clearly depend on the clinical expertise of the user and their facility in working with Isabel . A serious existential concern is whether presenting a clinician with dozens of diagnostic suggestions might be a distraction or lead to unnecessary testing . We have previously identified these trade-offs as an unavoidable cost of improving patient safety : The price of improving the odds of reaching a correct diagnosis is the extra time and resources consumed in using the CDSS and considering alternative diagnoses that might turn out to be irrelevant .14 In summary , the Isabel CDSS performed quickly and accurately in suggesting correct diagnoses for complex adult medicine cases . However , the test setting was artificial , and the CDSS should be evaluated in more natural environments for its potential to support clinical diagnosis and reduce the rate of diagnostic error in medicine .

O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O B I I I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O

clinical diagnosis support systems
isabel
internal medicine
diagnostic error
google
decision support


The

Chemistry

of

the

Postpharyngeal

Gland

of

Female

European

Beewolves

Females

of

the

European

beewolf

Philanthus

triangulum

possess

a

large

glove-shaped

gland

in

the

head

the

postpharyngeal

gland

-LRB-

PPG

-RRB-

They

apply

the

content

of

the

PPG

to

their

prey

paralyzed

honeybees

where

it

delays

fungal

infestation

Here

we

describe

the

chemical

composition

of

the

gland

by

using

combined

GC-MS

GC-FTIR

and

derivatization

The

PPG

of

beewolves

contains

mainly

long-chain

unsaturated

hydrocarbons

-LRB-

-RRB-

lower

amounts

of

saturated

hydrocarbons

-LRB-

-RRB-

and

minor

amounts

of

methyl-branched

hydrocarbons

-LRB-

-RRB-

Additionally

the

hexane-soluble

gland

content

is

comprised

of

small

amounts

of

an

unsaturated

alcohol

an

unknown

sesquiterpene

an

octadecenylmethylester

and

several

long-chain

saturated

-LRB-

-RRB-

and

unsaturated

-LRB-

-RRB-

ketones

some

of

which

have

not

yet

been

reported

as

natural

products

Surprisingly

we

found

a

dimorphism

with

regard

to

the

major

component

of

the

PPG

with

some

females

having

-LRB-

Z

-RRB-

whereas

others

have

-LRB-

Z

-RRB-

as

their

predominant

component

The

biological

relevance

of

the

compounds

for

the

prevention

of

fungal

growth

on

the

prey

and

the

significance

of

the

chemical

dimorphism

are

discussed

Introduction

Hymenoptera

possess

a

huge

variety

of

exocrine

glands

-LRB-

e.g.

Hölldobler

and

Wilson

-RRB-

The

chemistry

and

function

of

different

types

of

these

have

been

studied

for

a

number

of

social

species

whereas

comparatively

little

is

known

from

solitary

bees

and

wasps

Recently

a

postpharyngeal

gland

-LRB-

PPG

-RRB-

has

been

described

from

a

species

of

digger

wasp

-LRB-

Strohm

et

al.

-RRB-

the

European

beewolf

Philanthus

triangulum

Fabricius

-LRB-

Hymenoptera

Crabronidae

formerly

Sphecidae

Melo

-RRB-

The

occurrence

of

this

gland

is

surprising

since

the

PPG

was

assumed

to

be

restricted

to

ants

-LRB-

Hölldobler

and

Wilson

Schoeters

and

Billen

Lenoir

et

al.

-RRB-

where

it

functions

in

generating

the

colony

odor

-LRB-

e.g.

Hefetz

et

al.

Soroker

et

al.

Vienne

et

al.

Dahbi

et

al.

Lenoir

et

al.

Oldham

et

al.

Soroker

and

Hefetz

for

a

review

of

other

proposed

functions

see

Eelen

et

al.

-RRB-

In

beewolves

the

PPG

has

a

unique

function

in

protecting

the

larval

provisions

from

microbial

attack

-LRB-

Strohm

and

Linsenmair

Herzner

and

Strohm

Herzner

et

al.

-RRB-

Female

European

beewolves

hunt

and

paralyze

honeybees

bring

them

to

their

nest

burrow

and

provision

one

to

six

bees

in

a

brood

cell

as

larval

food

for

one

progeny

Due

to

the

humid

and

warm

conditions

in

the

brood

cell

the

highly

nutritive

provisions

are

prone

to

detrimental

microbial

attack

-LRB-

Strohm

and

Linsenmair

-RRB-

Early

fungus

infestation

inevitably

destroys

the

food

resources

and

larvae

are

killed

by

fungal

toxins

or

starve

to

death

Observations

in

special

cages

-LRB-

Strohm

and

Linsenmair

-RRB-

revealed

that

beewolf

females

intensively

lick

the

bodies

of

the

paralyzed

bees

and

apply

the

secretion

from

the

PPG

to

the

bees

surface

-LRB-

Strohm

and

Linsenmair

Herzner

et

al.

-RRB-

This

treatment

has

the

effect

of

delaying

fungus

growth

for

to

days

which

is

a

highly

relevant

effect

given

the

short

larval

feeding

period

of

only

to

days

The

primary

mechanism

of

this

delay

is

not

a

direct

chemical

effect

of

the

secretion

on

fungi

but

the

prevention

of

water

condensation

on

the

bees

that

in

turn

impairs

the

germination

and

growth

of

fungal

spores

-LRB-

Herzner

and

Strohm

-RRB-

Male

European

beewolves

also

have

a

PPG

that

is

even

larger

than

in

females

-LRB-

Herzner

et

al.

-RRB-

However

it

serves

as

a

reservoir

for

the

scent

marking

pheromone

that

males

apply

to

their

territories

to

attract

females

-LRB-

Kroiss

et

al.

-RRB-

Despite

the

advanced

understanding

of

the

function

of

the

secretion

of

the

PPG

of

female

European

beewolves

there

has

been

no

detailed

investigation

of

its

chemistry

Therefore

we

analyzed

the

chemical

composition

by

using

combined

gas

chromatography-mass

spectrometry

-LRB-

GC-MS

-RRB-

gas

chromatography-Fourier

transform

infrared

spectroscopy

-LRB-

GC-FTIR

-RRB-

fractionation

and

derivatizations

Materials

and

Methods

Sampling

Beewolf

females

were

taken

either

from

a

field

population

close

to

the

Biocenter

of

the

University

of

Würzburg

Germany

or

from

a

laboratory

population

kept

at

the

University

of

Würzburg

-LRB-

daughters

of

field

caught

females

-RRB-

They

were

all

mated

with

actively

provisioned

nests

with

honeybees

and

were

between

and

weeks

old

To

identify

the

chemicals

of

the

PPG

females

that

were

freshly

killed

by

were

decapitated

and

their

PPGs

were

removed

from

the

heads

by

grasping

the

hypopharynx

with

tweezers

and

gently

pulling

the

attached

gland

out

through

the

mouth

-LRB-

Strohm

et

al.

-RRB-

The

glands

were

immersed

immediately

in

ml

n-hexane

-LRB-

Fluka

Chemie

GmbH

Buchs

Switzerland

-RRB-

that

had

been

distilled

prior

to

use

For

the

identification

of

the

components

in

the

PPG

the

glands

of

four

females

were

pooled

This

enabled

us

to

identify

minor

components

that

were

not

reliably

detectable

in

the

extracts

of

individual

PPGs

In

order

to

have

an

easier

method

to

obtain

the

content

of

the

PPG

we

investigated

whether

extracts

of

whole

heads

differed

in

composition

from

dissected

glands

There

was

a

large

amount

of

hydrocarbons

in

the

PPG

and

extracts

of

entire

heads

were

identical

to

extracts

of

the

dissected

glands

There

were

no

additional

peaks

detectable

and

the

proportions

were

the

same

-LRB-

see

Herzner

et

al.

for

an

analogous

procedure

for

male

PPGs

-RRB-

Thus

as

an

easier

alternative

to

the

dissection

of

the

PPG

entire

heads

were

extracted

To

obtain

data

on

PPG

content

variability

we

analyzed

individual

extracts

made

from

the

heads

of

females

Heads

of

freshly

killed

females

were

cut

off

and

extracted

in

distilled

hexane

for

h

An

internal

standard

-LRB-

octadecane

Sigma

St

Louis

MO

USA

-RRB-

was

added

to

assess

the

absolute

amount

of

compounds

in

the

PPG

-LRB-

although

octadecane

could

be

detected

in

pooled

samples

it

was

not

detectable

in

individual

samples

and

was

therefore

employed

as

an

internal

standard

-RRB-

Identification

Identification

of

the

chemicals

was

accomplished

by

GC-MS

Head

extracts

were

fractionated

by

solid

phase

extraction

-LRB-

SPE

Chromabond

unmodified

silica

ml

mg

Macherey-Nagel

Düren

Germany

-RRB-

with

hexane

as

the

first

and

dichloromethane

as

the

second

eluent

to

separate

nonpolar

and

polar

fractions

Alkanes

were

characterized

by

comparison

of

mass

spectra

and

retention

indices

with

those

of

purchased

standard

alkanes

-LRB-

Aldrich

Deisenhofen

Germany

-RRB-

Corresponding

alkenes

were

tentatively

identified

by

their

typical

mass

spectra

their

retention

indices

and

-LRB-

depending

on

availability

-RRB-

with

commercially

available

-LRB-

Aldrich

-RRB-

and

synthesized

standards

Dimethyl

disulfide

-LRB-

DMDS

-RRB-

derivatization

was

carried

out

to

determine

the

position

of

double

bonds

according

to

the

method

of

Dunkelblum

et

al.

-LRB-

-RRB-

The

configurations

of

the

double

bonds

were

determined

by

using

GC-FTIR

-LRB-

Attygalle

-RRB-

Methyl-branched

alkanes

were

identified

by

using

MS

databases

and

diagnostic

ions

and

by

determining

retention

indices

-LRB-

Carlson

et

al.

-RRB-

Details

on

the

identification

of

polar

compounds

are

given

in

the

results

section.For

the

identification

of

the

hydrocarbons

we

used

a

Hewlett

Packard

HP

Series

GC

System

coupled

to

a

Hewlett

Packard

HP

Mass

Selective

Detector

-LRB-

Agilent

Technologies

Böblingen

Germany

-RRB-

The

GC

was

equipped

with

a

fused

silica

capillary

column

-LRB-

m

mm

i.d.

μm

film

thickness

J

W

Folsom

CA

USA

-RRB-

Temperature

was

programmed

from

°C

to

°C

with

a

°C

min

heating

rate

held

for

min

at

°C

Helium

was

used

as

carrier

gas

with

a

constant

flow

of

ml/min

Injection

was

carried

out

at

°C

in

the

splitless

mode

for

min

The

electron

impact

mass

spectra

-LRB-

EI-MS

-RRB-

were

recorded

with

an

ionization

voltage

of

eV

and

a

source

temperature

of

°C

The

software

ChemStation

-LRB-

Agilent

Technologies

-RRB-

for

windows

was

used

for

data

acquisition.GC-FTIR

spectra

were

obtained

by

using

an

HP

GC

-LRB-

Agilent

Technologies

-RRB-

coupled

to

an

FTS

Tracersystem

-LRB-

BioRad

Hercules

CA

USA

-RRB-

The

GC

was

equipped

with

the

same

column

as

described

above

Temperature

was

programmed

from

to

°C

with

a

°C

min

heating

rate

Helium

was

used

as

carrier

gas

with

a

constant

flow

of

ml/min

Injections

were

carried

out

by

using

a

split/splitless

injector

at

°C

in

the

splitless

mode

for

s.

Injection

volume

was

μl

IR

spectra

were

recorded

by

scanning

times

in

a

frequency

range

from

to

with

a

resolution

of

Data

system

was

a

Dell

Optiplex

with

BioRad

WinIR

Pro

-LRB-

Version

-RRB-

Tracer

Software

and

Sadtler

IRSearchMaster

Data

Handling

and

Statistical

Analysis

Because

the

relative

amounts

of

compounds

constitute

compositional

data

they

were

transformed

to

logcontrasts

according

to

Aitchison

-LRB-

-RRB-

Zij

-LRB-

Yij/g

-LRB-

Yj

-RRB-

-RRB-

where

Zij

is

the

standardized

peak

area

i

for

individual

j

Yij

is

the

peak

area

i

for

individual

j

and

g

-LRB-

Yj

-RRB-

is

the

geometric

mean

of

all

peaks

for

individual

j

prior

to

statistical

analysis

Several

peaks

had

to

be

combined

for

the

quantitative

analysis

of

individuals

because

they

were

not

always

clearly

separated

in

the

chromatograms

To

test

for

differences

in

the

proportions

of

components

between

groups

we

subjected

the

transformed

data

to

exact

tests

for

two

independent

groups

-LRB-

SPSS

SPSS

Inc.

-RRB-

Results

Identification

and

Analysis

of

Hydrocarbons

in

the

PPG

The

chromatograms

of

the

crude

hexane

extracts

of

the

PPG

showed

a

total

of

peaks

that

represented

different

compounds

-LRB-

Fig.

Table

-RRB-

The

mean

amount

of

all

compounds

was

μg

-LRB-

minimum

μg

maximum

μg

N

-RRB-

All

alkanes

as

well

as

-LRB-

Z

-RRB-

-LRB-

Z

-RRB-

and

-LRB-

Z

-RRB-

were

identified

by

comparison

of

mass

spectra

and

retention

indices

with

those

of

available

standards

The

location

and

configuration

of

double

bonds

of

other

alkenes

were

determined

with

DMDS

derivatives

and

GC-FTIR

data

-LRB-

band

at

cis

configuration

of

RCH

CHR

Attygalle

-RRB-

Some

components

could

however

not

be

completely

characterized

-LRB-

location

of

methyl

groups

or

double

bond

configuration

of

double

bond

-RRB-

due

to

the

small

amounts

in

the

extracts

One

component

could

not

be

identified

at

all

There

were

more

unsaturated

-LRB-

-RRB-

than

saturated

compounds

in

the

nonpolar

fraction

of

the

PPG

extract

from

beewolf

females

Fig.

ion

chromatograms

of

the

hexane

extract

of

the

PPG

from

individual

female

European

beewolves

Philanthus

triangulum

with

a

-LRB-

Z

-RRB-

-LRB-

-RRB-

and

b

-LRB-

Z

-RRB-

-LRB-

-RRB-

as

the

predominant

hydrocarbon

peak

Numbers

correspond

to

the

numbers

in

the

peak

list

-LRB-

Table

-RRB-

Some

compounds

listed

in

Table

were

present

in

quantities

too

low

to

be

visible

in

these

chromatogramsTable

of

compounds

in

the

postpharyngeal

gland

of

females

of

the

European

beewolf

Philanthus

triangulumPeak

no.RICompound

nameDiagnostic

MS

-LRB-

Z

-RRB-

296122100Heneicosanea296132200Docosanea310142267Δ-X-Tricosene322152273

-LRB-

Z

-RRB-

-9-Tricosenea322162286Δ-7-Tricosene322172300Tricosanea32418233811

336222400Tetracosanea338232436Δ-14-Tricosen-6-one99

-LRB-

Z

-RRB-

-9-Pentacosenea350262492Δ-Pentacosene350272500Pentacosanea35228253713

-LRB-

Z

-RRB-

-9-Hexacosene364332600Hexacosanea366342639Δ-16-Pentacosen-8-one127

-LRB-

Z

-RRB-

378412800Octacosanea394422840Δ-18-Heptacosen-10-one155

-LRB-

Z

-RRB-

-LRB-

Z

-RRB-

-LRB-

Z

-RRB-

retention

index

X

Y

position

of

the

methyl-group

or

double

bond

not

known

Δ

configuration

of

the

double-bond

not

known

NI

not

identifiedaIdentification

with

available

standards

Identification

of

the

Constituents

in

the

Polar

Fraction

The

polar

fraction

contained

a

group

of

uncommon

constituents

Hydrogenation

of

double

bonds

with

and

palladium

on

carbon

-LRB-

Attygalle

-RRB-

as

well

as

DMDS

adducts

were

used

to

obtain

sufficient

information

by

mass

spectrometry

to

identify

these

compounds

was

identified

by

comparison

with

an

already

published

mass

spectrum

-LRB-

Yasui

et

al.

-RRB-

The

mass

spectrum

of

this

compound

showed

a

molecular

ion

of

m/z

and

two

diagnostic

fragment

ions

at

m/z

for

-LSB-

CO

-RSB-

and

at

m/z

for

-LSB-

CO

-RSB-

indicating

the

carbonyl

position

at

After

hydrogenation

of

this

unsaturated

ketone

the

resulting

as

well

as

the

of

the

untreated

extract

showed

identical

mass

spectra

-LRB-

m/z

-LSB-

-RSB-

-LSB-

-RSB-

M

-RRB-

and

identical

retention

indices

-LRB-

-RRB-

with

that

already

published

by

Yasui

et

al.

-LRB-

-RRB-

was

characterized

by

its

diagnostic

masses

-LRB-

m/z

-LSB-

-RSB-

-LSB-

-RSB-

M

-RRB-

Also

hydrogenated

was

tentatively

identified

as

by

comparison

with

a

commercially

available

MS

library

-LRB-

NIST

Stein

et

al.

-RRB-

The

double

bond

positions

in

these

and

additional

unsaturated

ketones

were

determined

from

mass

spectra

of

DMDS

adducts

Additionally

three

unsaturated

and

one

saturated

ketone

were

tentatively

identified

as

and

The

corresponding

alcohol

was

tentatively

identified

by

its

diagnostic

masses

after

hydrogenation

of

the

polar

fraction

with

lithium

aluminum

hydride

-LRB-

Attygalle

Table

-RRB-

-LRB-

Z

-RRB-

was

characterized

by

using

a

commercially

available

standard

-LRB-

Aldrich

-RRB-

Finally

a

compound

with

a

typical

sesquiterpene

mass

spectrum

was

found

in

small

amounts

in

the

polar

fraction

of

the

extract

We

could

not

determine

the

type

of

functional

group

in

this

sesquiterpene

Chemical

Dimorphism

One

alkene

dominated

the

composition

of

the

PPG

and

surprisingly

this

major

compound

differed

among

individuals

-LRB-

Fig.

Table

-RRB-

Among

the

females

whose

PPG

content

was

analyzed

had

-LRB-

Z

-RRB-

and

eight

had

-LRB-

Z

-RRB-

as

their

major

peak

-LRB-

from

here

on

referred

to

as

the

and

respectively

-RRB-

The

frequency

of

the

two

types

differed

significantly

from

equality

-LRB-

χ2

df

P

-RRB-

By

using

exact

tests

for

independent

samples

to

assess

whether

the

proportions

of

other

components

were

also

affected

by

the

major

peak

we

revealed

that

of

the

peaks

differed

significantly

between

the

and

the

-LRB-

Table

-RRB-

Generally

the

had

larger

proportions

of

components

with

longer

chain

lengths

Fig.

distribution

-LRB-

population

of

N

-RRB-

of

individual

female

European

beewolves

Philanthus

triangulum

based

on

the

proportion

of

-LRB-

Z

-RRB-

in

their

PPG

secretion

-LRB-

values

transformed

to

logcontrasts

-LRB-

Aitchison

-RRB-

Zij

-LRB-

Yij/g

-LRB-

Yj

-RRB-

-RRB-

where

Zij

is

the

standardized

peak

area

i

for

individual

j

Yij

is

the

peak

area

i

for

individual

j

and

g

-LRB-

Yj

-RRB-

is

the

geometric

mean

of

all

peaks

for

individual

j

-RRB-

There

is

a

clear

bimodal

distribution

i.e.

some

individuals

have

a

large

proportion

of

-LRB-

Z

-RRB-

whereas

some

have

a

small

proportion

there

are

no

intermediate

typesTable

proportions

of

selected

hydrocarbons

and

ketones

from

the

postpharyngeal

glands

of

individual

female

European

beewolves

P.

triangulum

that

had

either

-LRB-

Z

-RRB-

-LRB-

-RRB-

or

-LRB-

Z

-RRB-

-LRB-

-RRB-

as

the

major

componentaCompound

values

-LRB-

Peak

no

in

Table

-RRB-

MeanSDMeanSDPent-HeptHeneicosane

-LRB-

-RRB-

Docosane

-LRB-

-RRB-

Tricosenes

-LRB-

-RRB-

Tricosane

-LRB-

-RRB-

Methyltricosanes

-LRB-

-RRB-

Tetracosene

-LRB-

-RRB-

Tetracosane

-LRB-

-RRB-

Pentacosenes

-LRB-

-RRB-

Pentacosane

-LRB-

-RRB-

Methylpentacosanes

-LRB-

-RRB-

Hexacosene

-LRB-

-RRB-

Hexacosane

-LRB-

-RRB-

0.050.030.050.020.000.06716-Pentacosen-8-one

-LRB-

-RRB-

Heptacosene

-LRB-

-RRB-

Heptacosane

-LRB-

-RRB-

Octacosane

-LRB-

-RRB-

0.030.030.030.020.000.18218-Heptacosen-10-one

-LRB-

-RRB-

Nonacosene

-LRB-

-RRB-

Nonacosane

-LRB-

-RRB-

Hentriacontene

-LRB-

-RRB-

Hentriacontane

-LRB-

-RRB-

aTable

entries

include

mean

and

one

standard

deviation

-LRB-

SD

-RRB-

of

the

proportion

-LRB-

-RRB-

of

the

components

for

females

of

the

-LRB-

N

-RRB-

and

of

the

-LRB-

N

-RRB-

as

well

as

the

difference

-LRB-

-RRB-

and

the

significance

level

for

the

difference

according

to

an

exact

test

-LRB-

P

significant

differences

in

bold

-RRB-

bDifferences

were

calculated

from

the

original

data

rounding

of

values

produced

some

rounding

error

Discussion

The

PPG

secretion

of

beewolf

females

contains

predominantly

unbranched

unsaturated

long

chain

hydrocarbons

-LRB-

mainly

either

or

-RRB-

smaller

amounts

of

saturated

hydrocarbons

-LRB-

-RRB-

and

small

or

trace

amounts

of

methylalkanes

-LRB-

-RRB-

unsaturated

ketones

-LRB-

-RRB-

saturated

ketones

-LRB-

-RRB-

a

sesquiterpene

an

unsaturated

methylester

and

an

unsaturated

alcohol

The

secretion

of

the

PPG

of

beewolf

females

is

involved

in

the

preservation

of

their

honeybee

prey

that

serves

as

larval

food

-LRB-

Herzner

and

Strohm

-RRB-

Females

apply

large

amounts

of

the

PPG

secretion

to

the

prey

prior

to

oviposition

-LRB-

Strohm

and

Linsenmair

Herzner

et

al.

-RRB-

The

prevalence

of

unsaturated

hydrocarbons

in

the

PPG

of

beewolf

females

is

probably

related

to

the

preservation

function

The

preservation

seems

to

be

mainly

accomplished

by

a

physical

mechanism

-LRB-

Herzner

and

Strohm

-RRB-

the

secretion

prevents

the

condensation

of

water

on

the

bees

and

in

this

way

renders

the

microclimatic

conditions

unsuitable

for

the

growth

of

fungi

Possibly

the

PPG

secretion

covers

structures

on

the

paralyzed

bee

that

would

otherwise

function

as

effective

nuclei

for

the

condensation

of

water

Scanning

electron

microscopy

revealed

that

the

PPG

secretion

forms

a

contiguous

layer

over

the

whole

surface

of

the

prey

-LRB-

Herzner

and

Strohm

-RRB-

A

hydrocarbon

mixture

of

predominately

alkenes

might

be

an

ideal

means

to

build

up

such

layers

because

at

the

temperatures

that

prevail

in

beewolf

brood

cells

the

unsaturated

hydrocarbons

might

be

in

a

more

or

less

liquid

state

and

can

be

spread

easily

over

the

bee

surface

The

composition

of

hydrocarbons

on

the

cuticle

of

paralyzed

honeybees

is

dramatically

modified

due

to

the

treatment

by

beewolf

females

-LRB-

Herzner

et

al.

b

-RRB-

Untreated

honeybees

and

most

other

insects

bear

predominantly

saturated

straight

or

branched

hydrocarbons

-LRB-

e.g.

Howard

and

Blomquist

Schmitt

et

al.

-RRB-

Less

frequently

alkenes

constitute

large

proportions

of

cuticular

hydrocarbons

-LRB-

e.g.

on

a

termite

Howard

et

al.

on

an

aphid

parasitoid

Liepert

and

Dettner

on

workers

of

European

hornets

Ruther

et

al.

on

stingless

bee

foragers

Abdalla

et

al.

on

diapausing

butterfly

pupae

Kaneko

and

Katagiri

or

on

nesting

females

of

the

burrowing

bee

Simmons

et

al.

-RRB-

The

reasons

for

the

prevalence

of

saturated

hydrocarbons

in

some

species

and

unsaturated

hydrocarbons

in

other

species

are

unknown

Possibly

the

physicochemical

properties

of

the

surface

can

be

adjusted

to

specific

requirements

by

a

particular

mixture

of

saturated

and

unsaturated

compounds

-LRB-

e.g.

Gibbs

and

Pomonis

Gibbs

-RRB-

In

addition

to

the

ubiquitous

alkanes

and

alkenes

that

we

found

in

the

PPG

we

also

identified

long

chain

unsaturated

ketones

some

of

which

had

not

yet

been

described

as

natural

products

Only

-LRB-

Z

-RRB-

and

had

previously

been

reported

as

components

of

a

contact

sex

pheromone

from

females

of

the

white-spotted

longicorn

beetle

Anoplophora

malasiaca

-LRB-

Yasui

et

al.

-RRB-

The

function

of

these

compounds

in

the

secretion

of

beewolf

females

is

unclear

Besides

a

not-completely

identified

sesquiterpene

-LRB-

Z

-RRB-

and

these

ketones

are

the

only

identified

components

in

the

PPG

of

beewolf

females

that

have

a

functional

group

They

might

be

likely

candidates

to

exhibit

an

antifungal

effect

However

in

bioassays

no

direct

antifungal

effect

of

the

PPG

content

could

be

detected

-LRB-

Herzner

et

al.

-RRB-

Male

European

beewolves

have

some

of

the

same

ketones

and

a

slightly

shorter

unsaturated

ketone

in

their

marking

secretion

-LRB-

Schmitt

et

al.

Kroiss

et

al.

-RRB-

Their

exact

function

in

males

is

also

unknown

-LRB-

see

below

-RRB-

The

estimated

amount

of

hydrocarbons

in

the

secretion

of

the

PPG

differed

considerably

among

females

This

might

be

due

to

differences

in

physiological

status

size

and

age

Interestingly

the

mean

-LRB-

approximately

μg

-RRB-

and

maximum

-LRB-

approximately

μg

-RRB-

amounts

match

the

quantities

found

on

honeybees

that

were

embalmed

with

the

PPG

secretion

by

beewolf

females

Bees

are

each

embalmed

with

approximately

μg

-LRB-

Herzner

et

al.

-RRB-

and

females

provision

on

average

three

bees

per

day

and

a

maximum

of

ten

bees

per

day

-LRB-

Strohm

and

Linsenmair

-RRB-

Thus

an

average

female

has

available

the

necessary

amount

of

PPG

secretion

for

the

embalming

of

the

average

number

of

bees

caught

on

day

Likewise

the

maximum

amount

found

in

some

individuals

would

be

sufficient

to

embalm

the

maximum

number

of

bees

that

the

most

successful

females

hunt

per

day

Moreover

according

to

a

three-dimensional

reconstruction

based

on

histological

sections

the

maximum

volume

of

the

gland

was

estimated

to

be

μl

-LRB-

Strohm

et

al.

-RRB-

The

comparatively

large

size

of

the

gland

is

explained

by

the

need

to

provide

enough

secretion

for

the

treatment

of

several

prey

items

per

day

The

supply

with

such

large

amounts

of

unsaturated

hydrocarbons

might

be

costly

and

might

represent

a

considerable

part

of

the

cost

of

parental

care

in

this

species

-LRB-

Strohm

and

Linsenmair

Strohm

and

Marliani

-RRB-

Remarkably

beewolf

females

show

a

striking

dimorphism

with

regard

to

the

chemical

composition

of

the

PPG

secretion

The

major

component

is

either

-LRB-

Z

-RRB-

or

-LRB-

Z

-RRB-

and

there

are

no

intermediate

individuals

Both

compounds

are

widespread

among

aculeate

Hymenoptera

and

other

insects

-LRB-

e.g.

Ruther

et

al.

Simmons

et

al.

-RRB-

Most

other

components

of

the

PPG

secretion

also

differ

between

the

two

morphs

in

that

tend

to

have

larger

proportions

of

long-chain

compounds

Possibly

the

whole

metabolism

of

hydrocarbons

is

adjusted

to

longer

chain

lengths

in

the

Why

females

have

either

-LRB-

Z

-RRB-

or

-LRB-

Z

-RRB-

as

the

major

compound

is

unclear

Preliminary

analyses

-LRB-

E.

Strohm

G.

Herzner

M.

Kaltenpoth

unpublished

data

-RRB-

suggest

that

there

is

no

effect

of

age

or

physiological

status

on

the

expression

of

the

major

component

One

proximate

explanation

could

be

that

conditions

during

development

differ

between

the

females

and

cause

differential

gene

activation

-LSB-

for

example

the

synthesis

of

-LRB-

Z

-RRB-

might

be

induced

by

high

temperatures

because

of

the

presumably

higher

melting

point

e.g.

Gibbs

et

al.

Rouault

et

al.

-RSB-

However

our

study

specimens

were

bred

under

identical

temperature

conditions

in

the

same

climate

chamber

with

the

same

diet

-LRB-

honeybees

from

the

same

population

-RRB-

and

nevertheless

showed

this

dimorphism

Furthermore

an

analysis

of

beewolf

females

from

different

populations

ranging

from

northern

Germany

to

the

southern

valleys

of

the

Alps

not

only

showed

both

types

of

females

but

also

revealed

that

they

occurred

in

similar

proportions

-LRB-

Strohm

et

al.

-RRB-

Together

with

the

lack

of

intermediate

individuals

this

might

suggest

that

the

dimorphism

has

a

genetic

basis

Such

a

dimorphism

would

have

to

be

balanced

because

otherwise

one

morph

would

disappear

at

least

from

some

populations

either

because

it

has

a

selective

disadvantage

or

because

of

genetic

drift

One

possible

explanation

for

a

balanced

dimorphism

is

a

spatial

difference

in

the

suitability

of

the

two

alleles

Such

spatial

heterogeneity

might

either

be

generated

by

differences

in

abiotic

or

biotic

conditions

There

are

specialized

cuckoo

wasps

that

enter

the

nests

and

oviposit

on

the

bees

-LRB-

Strohm

et

al.

-RRB-

These

wasps

seem

to

employ

chemical

mimicry

in

order

not

to

leave

traitorous

signs

in

the

nest

Most

notably

the

chemical

mimicry

only

refers

to

the

of

females

-LRB-

Strohm

et

al.

-RRB-

Thus

the

might

be

disadvantaged

with

regard

to

the

rate

of

parasitism

but

this

could

be

balanced

by

other

advantages

Again

this

hypothesis

is

weakened

by

the

fact

that

the

proportions

of

both

types

of

individuals

are

similar

over

a

wide

geographical

range

despite

probable

differences

in

the

abundance

of

the

cuckoo

wasps

Compared

to

the

content

of

the

PPG

of

males

of

the

European

beewolf

-LRB-

Schmitt

et

al.

Kroiss

et

al.

-RRB-

females

have

fewer

components

with

functional

groups

and

lower

proportions

thereof

whereas

the

composition

of

the

alkanes

and

alkenes

is

similar

The

function

of

the

secretion

of

the

male

PPG

is

also

quite

different

Males

use

this

secretion

to

scent

mark

their

territories

and

to

attract

receptive

females

-LRB-

Simon-Thomas

and

Poorter

Evans

and

O'Neill

Strohm

Strohm

and

Lechner

Schmitt

et

al.

-RRB-

The

marking

secretion

is

stored

in

and

delivered

from

the

PPG

-LRB-

Kroiss

et

al.

-RRB-

Therefore

the

compounds

with

functional

groups

may

play

a

role

in

the

attraction

of

females

-LRB-

Herzner

et

al.

Kroiss

et

al.

-RRB-

and

may

also

convey

some

additional

information

on

male

quality

and

suitability

as

a

mate

-LRB-

Herzner

et

al.

Kaltenpoth

and

Strohm

-RRB-

In

summary

females

of

the

European

beewolf

P.

triangulum

have

large

amounts

of

mainly

unsaturated

hydrocarbons

in

their

PPG

The

composition

of

the

secretion

is

probably

shaped

by

its

function

as

an

antifungal

coating

of

the

prey

paralyzed

honeybees

Thus

the

function

of

the

PPG

of

beewolf

females

differs

entirely

from

this

gland

in

ants

However

the

general

chemistry

is

consistent

with

that

found

in

ants

This

supports

earlier

arguments

based

on

morphology

ultrastructure

and

behavioral

context

-LRB-

Strohm

et

al.

-RRB-

that

the

PPGs

of

these

two

taxa

are

homologous

Comparison

of

physiological

aspects

of

the

PPGs

of

ants

and

beewolves

as

well

as

the

investigation

of

other

aculeate

Hymenoptera

is

necessary

to

obtain

further

insights

into

the

evolution

and

function

of

the

PPG

in

this

group

of

insects

O O O O B I O O O O O O O O O O B I O O O O O O O O O O O B I O B O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O B O O O O O O O O O O O O O O O O O O O O O B I O O O B O B O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O B O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B I O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O B O O O O O O O O O B O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O B O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B O O O O O O O O O O O O O B O O O O O O

antifungal
crabronidae
hymenoptera
philanthus triangulum
postpharyngeal gland
ppg
sphecidae