Feeding behavior, natural food, and nutritional relationships of larval mosquitoes.

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Annu. Rev. Entomol. 1992.37:349-374. Downloaded from www.annualreviews.org Access provided by University of Newcastle upon Tyne on 12/14/16. For personal use only.

Annu. Rev_ Enlomol. 1992. 37:349-76 Copyright © 1992 by Annual Reviews Inc. All righls reserved

FEEDING BEHAVIOR, NATURAL FOOD, AND NUTRITIONAL RELATIONSHIPS OF LARVAL MOSQUITOES R.

W. Merritt

Department of Entomology, Michigan State University, East Lansing, Michigan

48824 R. H.

Dadd

Department of Entomological Sciences, University of California, Berkeley, California

94720

E.

D. Walker

Department of Entomology, Michigan State University, East Lansing, Michigan

48824 KEY

WORDS:

Culicidae, mosquito larvae, feeding modes, feeding mechanisms, nutrients

PERSPECTIVES AND OVERVIEW

Mosquitoes (Diptera:Culicidae) are medically the most important group of insects, both in number of disease agents they transmit and the magnitude of health problems these diseases cause worldwide (128). Mosquito control 349 0066-4170/92/0101-0349$02.00


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MERRITT, DADD & WALKER

relies on materials whose efficient use requires knowledge of the behavior of the target species. Much of what we know about the detailed behavior of individual insect vectors resulted from observations made during the pre-DDT era of the 1920s and 1930s. The availability of synthetic insecticides reduced the need for vector behavior studies, because insecticides could be applied under a wide range of conditions and kill target insects on contact. Because of environmental considerations and the development of resistance (20), vector control has moved from the use of broad spectrum, persistent chemicals to more specific control materials, such as microbial insecticides, parasites, and pathogens. Several of the more recent mosquito biological control agents, such as bacteria [i.e. Bacillus thuringiensis var. israelensis (or Bti) and Bacillus sphaericus] and fungi (i.e. Lagenidium giganteum) must be ingested by larvae to be effective (22, 58, 89). Both species of bacilli are obligatory stomach poisons (10, 57), while zoospores of L. giganteum, once inside the larval mouth, generally penetrate the tissues of the digestive tract (98). The success of Bti and B. sphaericus as particulate larvicides is greatly enhanced by knowledge of: (a) the particle sizes that are ingested optimally by larval mosquitoes in their natural habitats and (b) the areas where the larvae forage (3, 54, 106, 147). In addition, recent cloning successes with genes coding for the endotoxins in Bti (10) suggest that new microbial insecticides could be engineered as mosquito control agents if such studies are melded with investigations of the food resources (e.g. other bacteria and algae) available to larvae in their natural habitat. Thus, research on larval mosquito feeding behavior and the nature of their diet has taken on increased signifi cance in recent years. Laboratory studies have suggested that vector competence (i.e. the internal physiological factors that govern the infection of human pathogens in a mosquito) (59) varies with the quality of the larval environment. Other studies indicated that larval stress, primarily caused by food limitation within habi tats, not only produced small adults, but adversely affected larval survival, developmental rates, and adult fitness (62, 70, 73, 110). For example, in some laboratory studies, smaller females (which reflects reduced larval food availability) were more competent vectors for arboviruses than larger females (60,68,115). An understanding of the spatial and temporal distribution of the dietary resources available to larval mosquitoes in their natural habitats could clarify the relationships among food availability, vector competence, and" mosquito fitness. Several recent treatises discuss larval mosquito food or the nature of the organic material available to aquatic invertebrates in different habitats (27, 87, 151). This review describes what is known about the feeding behavior, natural food, and nutritional relationships of different nonpredatory mosquito larvae. Feeding behavior encompasses mechanisms used in food acquisition, larval


FEEDING IN LARVAL MOSQUITOES

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distribution, posture, and orientation within habitats. Species of mosquitoes use different morphological structures and feeding modes (e.g. filtering, suspension feeding, browsing, interfacial feeding) in food acquisition; previ ous studies (54, 77, 116, 137) often cataloged different species as facultative or obligate feeders using the above, and several other, feeding modes. We propose a new classification of major feeding modes for nonpredatory mos quitoes, with the intention of clarifying previous definitions and bringing them into the general context of feeding terminology used with other aquatic invertebrates. FEEDING BEHAVIOR

Feeding Mode Definition The term feeding mode can be defined as how aquatic invertebrates acquire their food in nature. Feeding mode is synonymous with feeding type, feeding method, or feeding habit of previous authors (56, 77, 116, 137); however, we use the term habit to refer to how individuals maintain their location or existence (i.e. planktonic, clingers, etc) or move (i.e. swimmers, divers, etc) (104). Mosquito larvae, along with other aquatic invertebrates, have evolved morphological adaptations that allow behavioral flexibility for feeding on diverse resources (87, 147). Thus, several investigations have described different feeding modes and modifications of the larval mouth parts and their relationship to different modes (56, 77, 116, Ill, 120, 137).

Confusion Surrounding Current Feeding-Mode Classification Schemes

The meanings implied by the different feeding modes are somewhat vague, vary with the author, and hence are controversial. For example, Montschad sky (111) recognized two categories of mosquito larvae: vegetarians and predators, dividing vegetarians into surface feeders, plankton feeders, and substratum feeders. According to Harbach (71), Montschadsky imposed generic restrictions, placing all Anopheles species into the surface-feeder class, yet some of these species are known predators. Surtees' (137) classification scheme, the most widely accepted, recognized three categories: filter-feeders, browsers, and predators. Pucat (116) followed Surtees' (137) classification and, based on differences in mouthpart morphology of two species, found an intermediate feeding-type between filter-feeders and brows ers. Harbach (71) felt that the classification used by Montschadsky (111) more accurately delineated the feeding modes exhibited by culicid larvae than did that of Surtees (137), and he defined the following feeding categories: predators, plankton feeders, surface feeders, bottom feeders, and scavengers.


352


The latter two categories included the browsers of Surtees' (137) classifica tion. Harbach (71) also suggested that bottom feeding may be a specialized type of filter feeding. More recently, Dahl et al (56) substituted "suspension feeders" for the filter-feeders and "brushers" for the browsers of Surtees (137), Pucat (116), and Aly (4). Additional terms such as "interfacial" and "eddy" feeders of Renn (120), "chewers" and "biters" of Christophers (23), and "gnawers" of Beklemishev (17) have been used to describe particular feeding modes. Morphologically, a reduction in the numbers and lengths of lateral palatal brushes, maxillary setae, and mandibular setae (with an increase in the sclerotization, size, and serration of the mandibular teeth) generally occurs in progression from filter-feeders to predators (71, 116, 137). Montschadsky (111) and Surtees (137) proposed an evolutionary gradient from filter-feeders (plankton feeders), as the most primitive, to bottom feeders, scavengers, and predators, as the most advanced. Harbach (71), however, noted that the morphological data were insufficient to permit these conclusions and in dicated that scavengers were just as likely to be the primitive group that may have given rise to both plankton feeders and predators. Although each of the above evolutionary schemes were well conceived and plausible, most studies that have since contributed information have considered morphological aspects and extrapolated them to behavioral consequences. Few studies have simultaneously considered morphological adaptations with even qualitative, much less quantitative, behavioral observations. Differing opinions among investigators on feeding-mode classification schemes and nomenclature can be attributed to several factors. First, the morpho-behavioral mechanisms involved in food acquisition by larval mos quitoes have been poorly understood. Baseline studies on comparative func tional mouthpart morphology and proposed homologies have resulted in different interpretations of feeding modes because morphological structures (e.g. lateral palatal brushes, mandibular brushes and combs, maxillary brushes and filaments) and the physical dimensions of these structures vary among species and even within genera (97, 116, 118). To understand how feeding morphology and mechanisms are interrelated in Culicidae, a function al model of feeding behavior and its relation to fluid conditions is needed, similar to the recent comprehensive studies by Dahl et al (56) and Widahl (150) on suspension feeding and by Craig and coworkers on larval Simuliidae filter-feeding (31,34). In addition, confusion has resulted from authors using different mouthpart terminology for the same structures (116). Harbach & Knight (72) have contributed immensely to solving this dilemma by creating a taxonomist's glossary of mosquito anatomy for all life stages. Another factor contributing to different views on feeding-mode classifica tion is that, functionally, more than one feeding mode (e.g. filtering, brows-



353

ing) can occur in individuals of the same species. For example, Rasnitsyn & Yasyukevich (119) found that larvae of three different surface-feeding An opheles species also fed at water depths up to 8 cm. Merritt et al (107) observed that Coquillettidia perturbans larvae, a species that lives attached to submerged roots of freshwater macrophytes, had two feeding modes: suspen sion feeding in the water column and brushing from sediment and plant surfaces. Aedes triseriatus, a tree-hole inhabitant, demonstrated four feeding modes: suspension feeding, interfacial feeding, brushing, and chewing (144); cannibalism was also observed under laboratory conditions (83). In addition, several investigators have observed scavenging by Aedes aegypti larvae on carcasses of their own or related species (82, 100). The above findings cast doubt on the frequent assumption that species, let alone genera, can broadly be classified into such categories as obligate brushers, facultative suspension feeders, and so on (54, 149). A detailed analysis of behavioral patterns showed that larval feeding behavior was more flexible (144). We believe that mosquito species are rarely absolutely re stricted to a single feeding mode.

Variation in Feeding Modes Larval posture and movement during a feeding mode varies greatly. An opheles larvae in marsh habitats aggregate at air-water interfaces such as plant stems and algal mats ( 1 14 , 143) with their bodies floating parallel to the water surface (24). They then feed in an interfacial manner, their bodies usually remaining still if not disturbed. Although this aggregation at air-water-plant interfaces may represent an adaptation to avoid predation (29, 114), food resources are probably also more abundant at these interfaces. During suspension feeding (collecting-filtering), larvae belonging to genera other than Anopheles hang by their respiratory siphons from the water surface and may exhibit different and characteristic movements when changing posi tions (136). Larvae of some species (e.g. Culiseta inornata, Coquillettidia perturbans, Psorophora discolor) orient upside down, propped up on their antennae, at the bottom of habitats (56, 107, 136); this may allow suspension of settled particles from the substrate resulting from currents created by the mouth parts. Some Aedes larvae shift between different feeding modes during their normal behavioral activities (129). Aedes triseriatus fourth instars split their feeding time (90.8% of the total time budget) between submerged feeding and feeding near or at the water surface. The feeding modes exhibited while submerged and at the surface were linked by diving and rising be haviors, indicating that these mosquito larvae normally moved through a loop of feeding modes occurring at both positions. Aedes communis larvae also divide their time between surface and subsurface feeding modes; younger larvae devote comparatively less time to feeding underwater or at the bottom

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&

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than older instars (113). Fourth instars of Culex territans spend all of their time suspension feeding with their respiratory siphon attached to the air-water interface, while fourth-instar Culex torrentium occasionally leave this posi tion and feed in the water column or at the bottom substrate (113).

Proposed Classification of Feeding Modes Annu. Rev. Entomol. 1992.37:349-374. Downloaded from www.annualreviews.org Access provided by University of Newcastle upon Tyne on 12/14/16. For personal use only.

In

an

attempt to clarify feeding mode categories of

nonpredatory

larval

mosquitoes, we propose a classification based on the functional-feeding group concept, which has been applied to other freshwater organisms as well

(32, 33, 104, 151). This new classification uses information on larval culicid mouthpart morphology (27, 56, 71, 116, 120, 137). Functional feeding

groups are defined according to adaptations for food acquisition. Food resource categories (i.e. living or dead, plant or animal, in addition to nonnutritive materials such as silts and clays) have been chosen on the basis of: (a) particle size range (e.g. coarse or fine) of the potential food material and (b) its general location [e.g. tightly adhered to surfaces (periphyton) suspended in the water column, loosely deposited on surfaces or in sediments, in litter accumulations] (104). The mechanisms and morphological structures involved in filter- and suspension-feeding marine and freshwater invertebrates other than mosquitoes are reviewed elsewhere (80, 81, 108, 124, 147, 151). Our classification divides nonpredatory larval culicids into four behavioral feeding modes: (a) collecting-filtering, (b) collecting-gathering, (c) scraping, and (d) shredding (Table 1). Collectors are animals that have feeding methods, either passive or active, ad apted for gathering fine particulate ,

organic material as food. Passive methods for feeding on suspended particles depend on existing currents to bring food to the animal, while active methods

creating feeding currents (151). The collectors can be subdivided into those individuals that remove particles from the water column (suspension- or filtering-collectors) and those that remove particles deposited on surfaces or in crevices (gathering- or deposit-collectors). The involve energy expenditure in

collecting-filtering feeding mode can be defined as the removal of fine particulate organic material

(FPOM;

size range 0.45 /Lm to 1 mm) from

suspension, regardless of the filtering mechanism (147). This category con

sists of the filter-feeders of suspension-feeding zooplankton (140) and in cludes the following previously named feeding-mode categories: filter-feeders (4, 116, 137), interfacial and eddy feeders (120), plankton and surface feeders (71, 111), and suspension feeders (56, 113). Several genera of mosquitoes (e.g. Anopheles, Culex, Culiseta, Aedes, in part), occupying an array of different habitats from lake edges to artificial containers, normally exhibit the collecting-filtering feeding mode. The feed ing microhabitat of this group varies vertically from feeding at the air-water interface in most Anopheles species to feeding in the water column by Culex



355

and Culiseta and at plant root zones by Mansonia and Coquillettidia. The major food resource for these groups is microorganisms and detritus. In addition to mosquitoes, collecting-filtering is well documented in several other dipteran families and in the Ephemeroptera and Trichoptera (147). The collecting-gathering feeding mode can be defined as the resuspension of loosely attached (usually particles that do not have any type of hold-fast attachment structure) or deposited fine particulate organic material from surfaces or crevices or interstitial spaces by the action of the larva's lateral palatal brushes. This feeding mode is similar to the deposit-feeding of zoo plankton (151) and several aquatic insects such as chironomid midges (Chir onominae) and ephemerid mayflies (147). Among mosquitoes, numerous Aedes species use this feeding mode by swimming or diving to substrates in their habitats and resuspending the loosely attached material with their lateral palatal brushes, thus acquiring the names browsers and brushers, or scaveng ers and bottom feeders, of previous authors (4, 56, 71, 116, l 37, 149). Other mosquito genera (e.g. Haemagogus, Wyeomyia) also have collector-gatherer representatives, and these groups generally occupy natural containers, such as 1eaf axils, pitcher plants, and other phytotelmata, where microorganisms and detritus accumulate on plant surfaces (65, 93). The third behavioral feeding mode involves the removal of food that is tightly adhered to mineral and organic surfaces (e.g. periphyton), as opposed to loosely attached material with no special attachment structures of any kind. This feeding mode has been termed scraping, and some families of Trichop tera (e.g. G10ssosomatidae) and Ephemeroptera (e.g. Heptageniidae) have morpho-behavioral adaptations for shearing off (i.e. grazing upon) attached food, such as algae or stalked protozoans (33, 104). One species of mosquito, Aedes atropalpus, and surely others, scrapes periphyton off mineral sur faces in rockholes and pools (74) (Table 1). Some scavengers and bottom feeders of Harbach (71) also may utilize the scraping feeding mode, as he described mosquito larvae that ingested food by scraping up detritus or abrad ing material from the surface of submerged objects. Species exhibiting this mode probably also spend time in the filtering and/or gathering collec ting modes. The shredding feeding mode in larval mosquitoes consists of chewing, biting, or gnawing off small fragments from coarse particulate organic matter (CPOM; size range generally> 1 mm) such as leaves, filaments of macroal gae, or other plant parts. Yet these larvae may also feed off of dead in vertebrates, often of their own kind. Species with this feeding mode have been described by previous authors as chewers (23, 144), biters (87), and gnawers (17), or have been included under the scavenger and browser cate gory (71, 82, 116). Several species belonging to different genera have been observed using this feeding mode; however, scraping is not the predominant


Table 1

Summary of ecological data for nonpredatory Culcidae with emphasis on trophic relationships Trophic Relationships Predominant feeding

Feeding

Food

Selected

(examples)

Habitat"

Habit

modeb

microhabitat(s)

resource(s)

referencesc

Most Anopheles spp.

Lentic: lake edges, swamps

Hypo-neustonic;

Collecting-

Air-water in-

Microorganisms

18, 28, 75, 78,

Taxa

and marshes, shallow

planktonic

permanent ponds; lotic;

swimmers

filtering

terface

and detritus

86, 87, 120, 127, 145

flowing streamsdepositional areas

Culex spp. , Culiseta spp . , Orthopodomyia spp. , Uranotaenia spp . , Aedes spp. (in part)

Lentic: ponded streams,

Mainly planktonic

lake edges, swamps and

swimmers, some

marshes, shallow per-

divers

Collecting-

Water column

filtering

Microorganisms

15, 75, 78, 86,

and detritus

87,93, 116

Microorganisms,

67, 69, 101, 145

manent ponds, intermittent ephemeral puddles, natural (phytotelmata) and artificial containers, subterranean habitats

Coquillettidia spp . , Mansonia spp.

Lentic: swamps and marshes, shallow permanent

Clingers (stems and roots of plants).

ponds (on vascular hyd-

Piercing respira-

rophytes)

tory siphon

Collectingfiltering

Plant root zones

some detritus


Aedes spp. (in part) , Psorophora spp., Haemogogus spp., Wyeomyia spp.

Lentic: swamps and marshes, shallow temporary

Planktonic swimmers, divers

Collectinggathering

Surfaces, sediments, some

Microorganisms and detritus

ephemeral puddles, nat-

water column

ural containers (phy-

and at air-

totelmata), subterranean

water interface

75, 86, 87, 93, 116, 143-145

feeding in

pools, intermittent

3, 9, 19, 63-65,

habitats

Aedes atropalp us

Lentic: intermittent

Planktonic divers

Scraping

Mineral and

Microorganisms,

ephemeral puddles (rock-

organic sur-

mainly peri-

holes and pools), artifi-

faces

phyton (algae)

74; E. D.

Walke�

cial containers (tires)

Aedes triseriatus, Aedes aegypti, Culex bitaeniorhynchus group, Culiseta in ornata, Culiseta lon giareolata, Tripter oides spp. a

Lentic: ponded streams,

Planktonic swim-

Shredding

Organic surfaces,

Microorganisms

23, 27, 82, 86,

and detritus;

87, 116, 139a,

artificial containers (e.g.

parts of dead

141, 144

treeholes, tires, water

invertebrates

marshes , natural and

mers, divers

sediments

jars)

Habitat classification after Merritt

&

Cummins

(104)

and Laird

(87).

b Species examples refer to members of a particular genus that may use the noted behavioral feeding mode as part of their total repetorie, rather than exclusively (see text). e

Emphasis on trophic relationships.

d Unpublished observations.

VJ VI -...J

358

MERRITI, DADD & WALKER


mode for any of these and has only been recorded as part of a total repertoire (Table 1). Not included in this review and classification scheme of larval mosquito feeding modes are the obligatorily predatory Culicidae; however, in several instances, representatives of nonpredatory feeding modes may occasionally ingest live animals in the course of their feeding (83, 87).

Feeding Mechanisms Considerable research attention has focused on the organization of mouthpart actions involving (a) removal of suspended food particles from the water column or from surfaces, (b) entrapment of food, (c) manipulation or mastica tion within the preoral cavity, (d) ingestion into the true mouth, and (e) food bolus formation in the pharynx prior to swallowing (27, 118). One investiga tion involved a systems analysis of mouthpart components

(56).

The larval mouth parts (Figure 1) bear setae and spicul es configured into

brushes, combs, and sweepers, often referred to as mouth brushes. However, this term usually refers to the lateral palatal brushes (LPBs). These brushes, borne on the labrum, extend and retract in distinct phases owing to the contraction and relaxation of lateral labropalatal muscles, which attach to the tormae (To in Figure 1). During flexion, the filaments of the LPBs form filament rows because of the linear arrangement of the bases of the filaments in the lateral palatal penicular area (LPPA). The rows move through the water like the flipped pages of a book, creating flows of water that carry suspended or floating particles toward the head (56). An Anopheles larva rotates its head 180°, so that the LPBs are directed against the air-water interface while its body remains parallel to the interface (16, 24, 120). The beating of the LPBs occurs at a stroke frequency of approximately 5 cycles/s for Anopheles quadrimaculatus fourth instars at 21°C. During flexion, approximately 12 filament rows are formed that pro duce linear or slightly curvilinear currents in the water comprising the surface film. These currents converge at the midline of the head and are probably directed medially by the anteromedian palatal brush (APBr in Figure 1), which beats in opposite phase to the LPBs at the same stroke frequency (27, 126). Particles move along these currents in distinct starts and stops without any inertia, confirming that drag and high viscous forces at low Reynolds numbers govern particle movement in the currents. As particles approach the head, they accelerate as the flows narrow, and particles move between the LPBs, past the anteromedian palatal brush, and into the preoral cavity or feeding groove (FG). Simultaneously, water comprising the flows moves laterally and then downwards between the labrum and the antennae (A in Figure 1), forming a coherent vertical plume that may extend several centimeters into the water if feeding is continuous (120). This plume consists

LARVAL

MOSQUITOES

359


FEEDING IN

MnB

Figure 1

(Top) Scanning electron microscopy preparation of Culisera inornata fourth-instar

head, anteroventral view. (Bottom) Parasagittal section of Anopheles larval head, showing some mouthparts and associated structures [adapted after Harbach & Knight (72)]. Abbreviations: A, antenna; APBr, anteromedian palatal brush; CIR, ciypeolabrai ridge; Dm, dorsomentum; FG, feeding groove; Lh, labiohypopharynx; LPB, lateral palatal brush; LPPA, lateral palatal penicular area; LR, laciniorastrum; Mn, mandible; MnB, mandibular brush; MnS, mandibular sweeper; Mx, maxilla; MxB, maxillary brush; Pha, pharynx; SeS, sellar setae; To, torma; Vm, ven tromentum.


360

MERRITI, DADD & WALKER

of near-horizontal laminae of water mixed with uningested particles formed at each extension of the LPBs (R. W. Merritt, D. A. Craig, H. A. Vanderploeg, E. D. Walker & R. S. Wotton, in preparation). Besides interfacial feeding, Anopheles larvae also feed in a manner de scribed as eddy feeding, in which the movements of the LPBs create vortices laterally over each LPB, and particles caught in the vortices are ingested (16, 120). However, eddy feeding occurs at surface tensions lower than normally found in natural larval habitats (120). Analyses of the suspension-feeding system of mosquito larvae other than Anopheles (56, 149, 150) indicated that currents drawing particles from all directions towards the head were created by a combination of LPB strokes and contractions of the pharynx (Pha in Figure 1). Flexion of the LPBs caused in-flowing currents (150), while an outflow or ejection jet extended 10-40 mm from the larval head (56, 150). These counterflows, with Reynolds numbers estimated to be below 10, formed a toroid of moving water in which particles were entrained and carried to the pharynx (56, 150). The mechanisms by which food particles are actually captured and retained are poorly known. Studies of interfacial feeding by Anopheles larvae (16, 118, 120; R. W. Merritt, D. A. Craig, H. A. Vanderploeg, E. D. Walker & R. S. Wotton, in preparation) indicated that small particles are entrained in currents, but not intercepted directly by the LPB filaments. Dahl et al (56) have suggested that particles may be entrained in the boundary layers of the packages of waters formed by the oar-like strokes of rows of LPB filaments, but that they do not adhere to the filaments themselves. Consequently, the LPBs do not function as true filters, as do the labral brushes of black fly larvae (31). Schremmer (126) assumed that, in interfacial-feeding Anopheles larvae, food particles were first trapped either on filaments of the LPBs or on spicules (LR in Figure 1) of the maxillary brushes (MxB) and that the particles moved along two different routes among various mouthpart structures toward the pharynx. In the first route, particles that were trapped on the filaments of the LPBs were removed by the mandibular brushes. The particles then passed to the midpalatal brush, and from there they passed to the surface of the clypeopalatum. The mandibular sweepers (MnS) then shunted these particles into the pharynx, and presumably the sweepers were cleaned off by the dorsal and ventral fringes inside the pharynx. In the second route of particle move ment, particles were first intercepted by the maxillary brushes, removed by the mandibular rakes, and deposited on the denticles of the labiohypopharynx (Lh). These particles were then swept into the pharynx by occasional, deep movements of the maxillae (Mx) (126). In another interpretation (16), the mandibular incisors and molars of Anopheles larvae, combined with back ward movements of the hypopharynx, were thought to move food particles to the front of the mouth where they were then packed into the pharynx by the


FEEDING IN

LARVAL

MOSQUITOES

361

mandibular sweeper. Rashed & Mulla ( 1 18) rejected the participation of the maxillary brushes in Anopheles albimanus particle capture, but suggested that they served to "push and concentrate the particle flow in the mouth region ." In suspension-feeding larvae, the pharynx itself may provide the particle capture and retention mechanism through expansions that suck in particles brought to the feeding groove (i .e. preoral cavity) by the LPBs (23, 56). These particles are then sieved with the dorsal and ventral fringes of the pharynx, and excess water is pumped out with each pharyngeal contraction. In this model of particle retention, the mandibular and maxillary structures themselves are not thought to function as possible particle capturing structures (56). Capture of fine particles appeared to be enhanced by a mucosubstance secreted onto the LPBs (102), as in larval black flies (122). However, a follow-up histochemical study (55) found no mucus production beneath the LPBs, nor in or near the LPBs and lateral palatal plate, yet large agglomera tions of mucus stained positively in the feeding groove and pharynx. The origin of this mucus or the way that mucus helps particle entrainment in areas close to the larval mouth opening is not clear and further research is needed in this area. Large particles may also be caught up in currents created by the LPBs . In Anopheles larvae, these particles are crushed between the prementum and the mandibles ( 1 6, 120, 126). Rejected, large particles are discarded by a quick tum of the head after brief manipulation by the sellar setae (SeS), after which these particles sink (16, 120; R. W . Merritt, D. A. Craig, H. A. Vanderploeg, E. D. Walker & R. S . Wotton, in preparation). The food bolus forms in the pharynx, but the mechanisms for regulation of food bolus size are largely unknown (79). Dahl et al (56) found (by video observations) that Culiseta morsitans forms the food bolus in the sac-like ventral portion of the pharynx during strong contractions when excess water is expelled, before swallowing the food into the esophagus. However, Schrem mer (126) observed a separate contraction of the pharynx immediately before swallowing the bolus into the esophagus in Anopheles maculipennis. Forma tion of small food boluses was observed about every 4.4 s in A. quad rimaculatus fourth instars when particles were abundant (R. W. Merritt, D. A. Craig , H. A. Vanderploeg , E. D. Walker & R. S. Wotton, in preparation). These small boluses passed to the anterior esophagus and formed a larger bolus. The above discussion relates how little agreement there is on the precise mechanisms of particle capture. Most likely, different mechanisms coexist in every species. Similarly, the role of the pharynx and its fringes in filtering out or retaining particles before they are transported to the gut, and in the removal of excess water, is not clear. Possibly, different capture mechanisms apply for different particle sizes and densities in ambient water. Clarification of these

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WALKER

contentious matters awaits more detailed analysis using high-speed cinematography and videotaping (56, 150; R. W . Merritt, D. A. Craig, H. A. Vanderploeg, E. D. Walker & R . S . Wotton, in preparation) .


Regulation of Feeding The fact that mosquito larval feeding generally entails indiscriminate inges tion of seston does not mean that feeding by these larvae is uncontrolled. The intensity of feeding can be regulated by gustatory chemicals (such as organic solutes) that leach into ambient water from nutritious material, signaling a potential for good food in local places. The rate of beating of the LPBs does not change in response to gustatory phagostimulants , but rather the proportion of time spent beating. Larvae of Culex pipiens spend up to 30% of their time beating the LPBs, whether or not particles are present. In one study, introduction of a phagosti mulant solute induced nearly continuous movements of the LPBs for up to an hour (36). If particulate material was present , the larvae ingested it through the gut at a rate proportional to the cumulative time spent feeding; this movement depended entirely on whether larvae ingested additional solids . The rates of ingestion by larvae of Culex tarsalis, A. aegypti. and A. albima nus were four to six times faster when they were fed particulate food material than when provided with nonnutritive particles, which indicates the role of phagostimulation by chemical compounds contained in the food (117). Investigators tested the phagostimulant properties of a wide variety of solutes likely to act as cues for food (35, 50, 52). Findings from these studies showed significant phagostimulation for various crude, mixed nutrient materi als, such as yeast extract and whole nucleic acids. However, only adenylic acid and certain other nucleotides and nucleosides strongly stimulated a feeding response. Most single amino acids , sugars , salts, or vitamins did not stimulate a response. Synthetic diets (mixes of simple nutrients) were power ful phagostimulants even when lacking components such as nucleotides , suggesting that the additive effects of many weak stimuli could be important in inducing feeding. Similar phagostimulant effects on ingestion were found for larvae of Anopheles albimanus (7), Aedes vexans (5), A. aegypti, A. sierrensis. and C. tarsalis (R. H. Dadd, unpublished) . Such observations suggest that regulation of feeding activity by phagostimulants emanating from nutrient organic material is probably a general feature of mosquito larval behavior that may tend to optimize feeding in food-rich foci in the habitat . There is some evidence that mosquito larvae actively seek out or become aggregated at food-rich foci. A. vexans larvae dove more frequently upon depletion of particulate food suspended in the water column and congregated at the bottom when pellets of fish meal or yeast were available, but not when kaolin or calcined earth pellets of nonnutritive content were provided (8).

FEEDING IN

LARVAL MOSQUITOES

Using a choice arena, the attraction of various chemical baits for C.

quefasciatus

363 quin

was studied following the observation of apparent attraction to

certain mucilaginous seeds

(25). Larvae aggregated at baits containing nucleic ( 1 3, 6 1 ) and were similarly attracted strongly to less strongly to single amino acids ( 1 4).

acid or certain nucleotides casein hydrolysate, but

Nucleic acids and nucleotides are essential nutrients for mosquitoes and are


significant phagostimulants

(6).

They also are constituents of all living tissue,

and indeed are sometimes measured in natural waters as an index of general biomass

( 1 46).

Thus, they provide an excellent food cue for an omnivore.

Interestingly enough, for adult mosquitoes, adenylic acid and other nucleo tides are the principal phagostimulant that, in the female, controls engorge ment of the blood meal directly to the midgut

(66).

NATURAL FOOD AND RESOURCE UTILIZATION Gut Contents and Food Availability Microorganisms and particulate organic detritus generally constitute the major part of larval mosquito diets

(9, 27, 87, 145). Although most reports suggest

that mosquito larvae are not very discriminatory in the types of food they ingest, laboratory studies indicate that limited sizes of particles are ingested This selection may be a result of morphological and be

(37, 1 1 6, 147).

havioral adaptations for acquiring food

(71, 107, 1 1 6, 1 18), instar-specific (37). The majority of larvae ingest particulate matter that ranges from colloid-sized to 50 /Lm, and most studies report ingestion of a higher proportion of smaller particles (37, 101, 1 06). Smaller-sized particles, with high surface area:volume ratios, are more densely colonized by microorganisms than larger particles (33, 1 05) and

features

( 1 0 1 , 1 06),

and particle shape and texture

are an important factor influencing growth and population density of other filter-feeding aquatic insects

(21 , 1 47).

However, whether microorganisms

form the principal assimilated food item or whether other components of these fine particles are assimilated instead is unclear. The observations that solid material of any sort passes through the larval gut in feeding

(36)

0.5- 1.0

h during normal

indicate that probably no substantial resident gut fauna exists in

the ectoperitrophic space. According to

Dahl (54), the upper size limit for particles ingested depends

more on their density, and not necessarily on well-defined size classes. Her observations showed that low density and texture of particles might be a more important factor for ingestion than absolute size ranges, because flat, floccu lent, less dense particles

(-100

/Lm) have been observed to be ingested by

fourth instars. These large particles, in contrast to heavy particles, are more buoyant and would remain longer in suspension, allowing more time for possible capture.


364


Whether larvae were selective with respect to their food choice in nature was controversial in early studies (90, 125). Laird (87) suggested that food availability and larval feeding habits were limiting in terms of what was ingested. The literature contains no consensus as to whether the distribution of anopheline larvae in their habitats was a function of food availability or location (18, 96), and researchers have also debated the suggestion that abundance of microorganisms was a limiting factor for anopheline growth in natural habitats (75). For example, by eliminating algae in a small pond with copper sulfate, Coggleshall (28) demonstrated its absence in the food of A. quadrimaculatus. However, after recolonization in the same pond, algal cells were again observed in the larval gut. The particulate food materials comprising the gut contents of mosquito larvae can be examined using microscopic methods. This process of gut analysis (9, 145) omits colloidal or dissolved food materials that cannot be detected microscopically, yet such materials may be important in larval nutrition ( 17, 76, 1 30, 1 5 1). Most of the early studies on gut analysis concerned the types of food items found in anopheline larval guts (84, 1 09), but some also examined culicine guts ( 1 2, 78). Laird (87) broadly concluded that larval food consists of a range of living and nonliving materials, such as dust, bacteria (including cyanobac teria), unicellular algae (including zooflagellates), other protozoans, filamentous algae, and small metazoans (i.e . rotifers and crustaceans). Sever al studies have provided long lists of identified taxa of algae and other microorganisms found in larval guts (9, 18, 28, 127). Hinman (75) dissected the guts from larvae of several species in five genera and observed cyanobac teria, numerous unicellular and filamentous algae, zooflagellates and other protozoans, rotifers, crustaceans, organic debris, unspecified inorganic mate rials, spores, and insect scales. Perhaps the most thorough gut analysis of mosquito larvae was conducted by Howland (78), who dissected over 1 000 larvae of 8 species, identified the algae present, and ranked them by abund ance in the food. She concluded that the abundance of algae in the larval food was correlated with algal abundance in the habitats, and that culicines con sumed more algae than anophelines. Bacteria have been considered the most important of the microorganisms that comprise the food of mosquito larvae (23, 86, 87), and mosquito growth can occur on cultures of bacteria alone (76, 1 23). However, investigators were not able to visualize bacteria in dissections for purposes of gut analysis, possibly leading to the conclusion by many researchers that the majority of the food observed could not be identified beyond "indeterminate brown amorphous matter" ( 1 27). Consequently, the relative abundance of bacteria in relation to other particulate food items was not known. Recently, DNA binding fluorochromes were used in combination with epifluorescence mi-



365

croscopy to categorize and enumerate bacteria in the larval gut of mosquitoes. Using acridine orange, Nilsson (113) found in fourth instars of some Aedes, Culex, Culiseta, and Anopheles species that the number of bacteria per gut (i.e. per food bolus) ranged from 6.7 x 106 to 2.2 X 107. Using acridine orange, Marten (95) visualized bacteria but presented no data on numbers of bacteria in the food of Aedes albopictus larvae. Walker et al (145) used a different fluorochrome, 4'6-diamidino-2-phenylindole (DAPI), and differen tiated cocci, rods, and spirochetes in Aedes triseriatus, Anopheles quad rimacuiatus, and Coquillettidia perturbans fourth instars. The mean number of bacteria in the larval food bolus of these species was, respectively, 2.2 x 106,2.0 X 106, and 0.9 X 106. Other microorganisms and particles of detritus were also enumerated using the same slide preparations that were used for enumeration of bacteria; A. triseriatus (an inhabitant of containers) lacked algae completely, while bacteria were several orders of magnitude more abundant than algae or protozoans in the two other species. Merritt et al (107) conducted a more extensive gut analysis of C. perturbans larvae using DAPI and found that bacteria (cocci, rods, spirochetes,purple bacteria, and cyano bacteria), detritus particles, euglenoid protozoans, desmids, and diatoms comprised the majority of particulate food, in order of abundance. Fourth instars had a greater proportion of euglenoids, desmids, and diatoms than did the younger larvae. Variation in food abundance in larval habitats has been studied mostly in container habitats. Feeding by mosquito larvae reduced abundance of bacteria and protists in bamboo stumps (85) and regulated diversity and abundance of protists in Heliconia and Serracenia pitcher plants (1, 94). Rainfall diluted bacterial concentrations in the tree-hole habitats of A. triseriatus (143), while feeding by larvae and the inputs of stemflow water from trees reduced bacterial concentrations in field microcosms (142). Aedes sierrensis, another tree-hole inhabitant, utilized trophozoites of its own parasite, Lambornella clarki, as a food resource, and numbers of this parasite in tree-hole water were reduced in part as a result of mosquito feeding activity (148). Reductions in numbers of container-inhabiting Aedes and Culex larvae by predators general ly caused an increase in larval food (bacteria, protists, fungi, and rotifers) because of decreased mosquito feeding (121). However, this predation some times caused paradoxical decreases in potential mosquito food when other organisms (such as ostracods) were released from competition for food re sources with mosquito larvae (121). Howland (78), Ameen & Iversen (9), and Laird (87) all proposed that the ecological succession of microorganisms in larval habitats directly affects what the larvae ingest, through direct availabil ity. Laird (86, 87) has commented that a drawback of gut analysis is that many of the more fragile ciliates and other protists are rapidly digested and con-


366

MERRITI,

DADD & WALKER

sequently cannot be observed in dissections, even though they may comprise an important fraction of the ingested food. Indeed, Laird (87) further stated that intact algae observed in dissections of larval mosquito food may possibly represent nonfood items, as they may pass through the gut undigested and perhaps remain viable. However, algae may produce exudates that can be used by mosquito larvae (152). Laird (88) recently discussed the dilemma that surrounds the systematic position of various aquatic microorganisms, particu larly protozoans and blue green algae. He also gave an example of how very different interpretations of gut content analyses in mosquitoes can occur, based on the use of different microorganism-classification systems.

Nutritional Relationships For the past 50 years, the nutrients needed for larval growth and development of mosquitoes to reproductive maturity have been investigated in experiments using defined artificial dietary media. Earliest studies dealt almost exclusively with A. aegypti, and mainly used semisynthetic larval dietary media in which amino nitrogen was provided as a whole protein, such as casein. Even when dietary media with pure amino acids in place of whole protein were ultimately developed for A. aegypti (91, 131), other crude components were still re tained. Thus, this early work established most nutrient requirements quali tatively, but the nature of the nucleic acid and lipid needs remained ambiguous. Purer chemicals allowed holidic (fully defined) synthetic diets of more stringent definition to be used with C. pipiens (46). Specific nucleotides replaced nucleic acid (40). A beneficial effect of phospholipids (47), similar to that found in some of the old work with A. aegypti, was shown in C. pipiens to result from a need for a 20-carbon polyunsaturated, essential fatty acid related to arachidonic or eicosapentenoic acids (41). Phospholipid in the dietary medium was also necessary for the proper utilization of various phytosterols (48). These holidic diets allowed almost normal rates, with good survival, of development of C. pipiens to adults capable of protracted flight, and also supported good development of six other mosquito species (42). From these nutritional studies, certain features influencing the capability of naturally occurring foods to support mosquito larval development may be inferred. Table 2 lists classes of nutrients and other ingredients in synthetic diets used successfully with C. pipiens and the other species. All ingredients were in solution, so such diets lacked particulate solids, and ingestion was . entirely a matter of bulk drinking rather than particle separation and concen tration. A full range of minerals and water-soluble B vitamins, including choline, were needed. Like most Diptera, mosquitoes require nucleic acid components in the diet. Besides the usual 10 essential amino acids required by all insects lacking symbiotes (43), A. aegypti, C. pipiens, and C. tarsalis


Table

2

Composition of holidic synthetic dietary medium for mosquito larvae

Ingredients

Concentration

Nutrient status or

% W/Va

other function

Potential sources of nutrients

18 amino acids

1 . 50

1 1 or 12b essential

All organisms, mostly as protein

Sugar: maltose, sucrose, glucose, mixed

0.75

Beneficial

All organisms, mostly as storage carbohydrate

10 mineral ions

0 . 33

9 essential

All organisms, also silt

8 water-soluble vitamins (includes choline) 5 nucIeotides

Larval food quantity affects the capacity of adult mosquitoes to transmit human malaria.

Ejection of malaria sporozoites by feeding mosquitoes.

Drosophila adult and larval pheromones modulate larval food choice.

Relationships between feeding problems, behavioral characteristics and nutritional quality in children with ASD.

Capacity of mosquitoes to transmit malaria depends on larval environment.

Ovipositional Behavior of Anopheles gambiae Mosquitoes.

Shelter-building behavior and natural history of two pyralid caterpillars feeding on Piper stipulaceum.

Global perspectives on nutritional knowledge, eating behavior, household food security, and hunger.

Toxic Effect of Blood Feeding in Male Mosquitoes.

Coping with food-limited conditions: feeding behavior, temperature preference, and nocturnal hypothermia in pigeons.

How Nectar-Feeding Bats Localize their Food: Echolocation Behavior of Leptonycteris yerbabuenae Approaching Cactus Flowers.

Functional analysis of the larval feeding circuit in Drosophila.

Nutritional Intervention and Breakfast Behavior of Kindergartens.

Fauna and Larval Habitats of Mosquitoes (Diptera: Culicidae) of West Azerbaijan Province, Northwestern Iran.

Expression and light-triggered movement of rhodopsins in the larval visual system of mosquitoes.

Food selection in larval fruit flies: dynamics and effects on larval development.

Nutritional aspects and diets in food allergy.

Nutritional Guidelines and Fermented Food Frameworks.

The Norwegian food and nutritional policy.

Studies on the feeding of larval Argas persicus Oken.

Fauna and Larval Habitat Characteristics of Mosquitoes in Neka County, Northern Iran.

Eye color and feeding behavior of animals.

Sex-dependent effects of larval food stress on adult performance under semi-natural conditions: only a matter of size?

Natural variability in Drosophila larval and pupal NaCl tolerance.